Hepatitis E Virus Infection in Voluntary Blood Donors in the Russian Federation.

Transfusion-transmitted hepatitis E virus (HEV) infection is an increasing concern in many countries. We investigated the detection rate of HEV viremia in blood donors in Russia. A total of 20,405 regular repetitive voluntary non-renumerated blood donors from two regions (Moscow and Belgorod) were screened for HEV RNA using the cobas® HEV test in mini-pools of six plasma samples. Samples from each reactive pool were tested individually. The average HEV RNA prevalence was 0.024% (95% CI: 0.01-0.05%), or 1 case per 4081 donations. No statistically significant differences in HEV RNA prevalence were observed between the two study regions. The PCR threshold cycle (Ct) values ranged from 25.0 to 40.5 in reactive pools, and from 20.9 to 41.4 in reactive plasma samples when tested individually. The HEV viremic donors had different antibody patterns. Two donor samples were reactive for both anti-HEV IgM and IgG antibodies, one sample was reactive for anti-HEV IgM and negative for anti-HEV IgG, and two samples were seronegative. At follow-up testing 6 months later, on average, four donors available for follow-up had become negative for HEV RNA and positive for anti-HEV IgG. The HEV ORF2 sequence belonging to HEV-3 sub-genotype 3a was obtained from one donor sample. The sequencing failed in the other four samples from viremic donors, presumably due to the low viral load. In conclusion, the HEV RNA detection rate in blood donors in Russia corresponds with data from other European countries, including those that implemented universal donor HEV screening. These data support the implementation of HEV RNA donor screening to reduce the risk of transfusion-transmitted HEV infection in Russia.

Epidemiology of Viral Hepatitis in the Indigenous Populations of the Arctic Zone of the Republic of Sakha (Yakutia).

The indigenous populations of the Arctic regions of Russia experience the lowest coverage of health-related services. We assessed the prevalence of hepatitis A, B, C, D and E viruses (HAV, HBV, HCV, HDV and HEV) among 367 healthy adult Native people of the Arctic zone of Yakutia. The HAV seroprevalence was above and increased with age. The anti-HEV IgM and IgG antibody detection rates were 4.1% and 2.5%, respectively. The average HBsAg detection rate was 4.6%, with no positive cases identified in participants aged under 30 years, confirming the effectiveness of the newborn vaccination program that began in 1998. Anti-HDV antibodies were detected in 29.4% of HBsAg-positive cases. The anti-HCV and HCV RNA detection rates peaked in the age cohort of 50-59 years (10.8% and 3.9%). No statistically significant gender differences in the prevalence of different viral hepatitis were observed. The time-scaled phylogenetic analysis demonstrated that all HBV genotype A and D strains isolated in this study were autochthonous and had an estimated most common recent ancestor (MCRA) age of around the 11th to 14th century. Unlike HBV, the HCV strains of subtypes 1b, 2a and 2k/1b were introduced from other regions of Russia in the 1980s and 1990s. The HCV 1b sequence analysis revealed a series of transmission events. In conclusion, these data emphasize the urgent need for expanded viral hepatitis screening and care programs in the indigenous populations of the Arctic zone of Yakutia.

Post-Vaccination and Post-Infection Immunity to the Hepatitis B Virus and Circulation of Immune-Escape Variants in the Russian Federation 20 Years after the Start of Mass Vaccination.

A neonatal vaccination against the Hepatitis B virus (HBV) infection was initiated in Russia 20 years ago, with catch-up immunization for adolescents and adults under the age of 60 years launched in 2006. Here, we have assessed the humoral immunity to HBV in different regions of Russia, as well as the infection frequency following 20 years of a nationwide vaccination campaign. We have also evaluated the role of immune-escape variants in continuing HBV circulation. A total of 36,149 healthy volunteers from nine regions spanning the Russian Federation from west to east were tested for HBV surface antigen (HBsAg), antibodies to HBV capsid protein (anti-HBc), and antibodies to HBsAg (anti-HBs). HBV sequences from 481 chronic Hepatitis B patients collected from 2018-2022 were analyzed for HBsAg immune-escape variants, compared with 205 sequences obtained prior to 2010. Overall, the HBsAg detection rate was 0.8%, with this level significantly exceeded only in one study region, the Republic of Dagestan (2.4%, p < 0.0001). Among the generation vaccinated at birth, the average HBsAg detection rate was below 0.3%, ranging from 0% to 0.7% depending on the region. The anti-HBc detection rate in subjects under 20 years was 7.4%, indicating ongoing HBV circulation. The overall proportion of participants under 20 years with vaccine-induced HBV immunity (anti-HBs positive, anti-HBc negative) was 41.7% but below 10% in the Tuva Republic and below 25% in the Sverdlovsk and Kaliningrad regions. The overall prevalence of immune-escape HBsAg variants was 25.2% in sequences obtained from 2018-2022, similar to the prevalence of 25.8% in sequences collected prior to 2010 (p > 0.05). The population dynamics of immune-escape variants predicted by Bayesian analysis have remained stable over the last 20 years, indicating the absence of vaccine-driven positive selection. In contrast, the wild-type HBV population size experienced a rapid decrease starting in the mid-1990s, following the introduction of mass immunization, but it subsequently began to recover, reaching pre-vaccination levels by 2020. Taken together, these data indicate that it is gaps in vaccination, and not virus evolution, that may be responsible for the continued virus circulation despite 20 years of mass vaccination.

Silent HDV epidemics culminates in high levels of liver cirrhosis in endemic region despite 20 years of HBV vaccination.

The hepatitis delta virus (HDV) is believed to be a vanishing infection in countries with successful hepatitis B virus (HBV) vaccination programs. We assessed the current status of HDV infection in Tuva, a region of the Russia that has been highly endemic for HBV. The proportion of HDV-infected patients among HBsAg-positive patients in the regional registry in 2020 was 32.7% (786/2401). An analysis of the medical records of 514 HDV patients demonstrated that 37.5% (193/514) had liver cirrhosis at the first doctor's visit, and 7.4% of patients lived in families where another family member had HDV. All HDV patients were infected with genotype HDV-1, 94.5% had HBV genotype D, and 5.5% had genotype A. A serosurvey conducted among 1170 healthy volunteers showed that the average detection rate of HBsAg with anti-HDV was 1.0% (95% CI: 0.57-1.81%). No anti-HDV positive samples were detected in participants aged under 30 years. The HBsAg/anti-HDV positivity rate peaked at 7.4% in patients aged 50-59 years, which was significantly higher than in a similar age cohort surveyed in 2008 (1.6%, p < .0001). A Bayesian analysis showed that HDV circulation in Tuva resulted from two waves of introduction, the first in the 1810s (95% HPD: 1741-1834) from Central Asia, and the second in the 1960s (95% HPD: 1953-1979) from Russia. HBV has a much longer history of circulation in Tuva with the MRCA for the predominant genotype HBV-D dated to 972 (95% HPD: 535-1253) for subtype D1, 1274 (95% HPD: 936-1384) for D2, and 1173 (95% HPD: 1005-1618) for D3. A SkyGrid reconstruction of population dynamics showed an increase in the intensity of HDV spread in recent decades. This situation shows the need for HDV screening and prevention measures among people living with HBV.

Dynamic Changes in Hepatitis A Immunity in Regions with Different Vaccination Strategies and Different Vaccination Coverage.

The data on hepatitis A virus (HAV) seroprevalence are critical for the implementation of a universal mass vaccination (UMV) strategy. The latter has not been implemented in Russia; however, regional child vaccination programs have been adopted in some parts of the country. The aim of this study is to assess changes in HAV immunity within the last decade in regions of Russia with different vaccination strategies and different vaccination coverage rates. In regions where UMV has not been implemented and HAV vaccination coverage rates do not exceed the national average, the 50% seroprevalence threshold has shifted in the Moscow region from people aged under 40 years in 2008 to people aged over 59 years in 2020, and from people aged under 30 years to people aged over 40 years in the Khabarovsk region. In two regions (Yakutia and Sverdlovsk), a two-dose-based UMV scheme has been in place since 2011 and 2003, respectively, and in Tuva single-dose child immunization was launched in 2012. These regional programs have resulted in a significant increase in HAV seroprevalence in children and adolescents. In Yakutia, 50% herd immunity had been achieved by 2020 in age groups under 20 years, compared to 20−30% seroprevalence rates in 2008. In the Sverdlovsk region, HAV immunity has increased to >65% over the decade in children aged over 10 years, adolescents and young adults, whereas it declined in older age groups. However, a three-fold drop in HAV immunity has occurred in children under 10 years of age, reflecting a significant decline in vaccination coverage. In Tuva, HAV immunity rates in children under 10 years old increased two-fold to exceed 50% by 2020. These data suggest that UMV should be implemented on a national level. Measures to control vaccination coverage and catch-up vaccination campaigns are recommended in order to maintain the effectiveness of existing HAV vaccination programs.

Different evolutionary dynamics of hepatitis B virus genotypes A and D, and hepatitis D virus genotypes 1 and 2 in an endemic area of Yakutia, Russia.

BACKGROUND: The geographic distribution of the hepatitis B virus (HBV) and the hepatitis D virus (HDV) genotypes is uneven. We reconstructed the temporal evolution of HBV and HDV in Yakutia, one of the regions of Russia most affected by HBV and HDV, in an attempt to understand the possible mechanisms that led to unusual for Russia pattern of viral genotypes and to identify current distribution trends. METHODS: HBV and HDV genotypes were determined in sera collected in 2018-2019 in Yakutia from randomly selected 140 patients with HBV monoinfection and 59 patients with HBV/HDV. Total 86 HBV and 88 HDV genomic sequences isolated in Yakutia between 1997 and 2019 were subjected to phylodynamic and philogeographic Bayesian analysis using BEAST v1.10.4 software package. Bayesian SkyGrid reconstruction and Birth-Death Skyline analysis were applied to estimate HBV and HDV population dynamics. RESULTS: Currently, HBV-A and HDV-D genotypes are prevalent in Yakutia, in both monoinfected and HDV-coinfected patients. Bayesian analysis has shown that the high prevalence of HBV-A in Yakutia, which is not typical for Russia, initially emerged after the genotype was introduced from Eastern Europe in the fifteenth century (around 600 (95% HPD: 50-715) years ago). The acute hepatitis B epidemics in the 1990s in Yakutia were largely associated with this particular genotype, as indicated by temporal changes in HBV-A population dynamics. HBV-D had a longer history in Yakutia and demonstrated stable population dynamics, indicating ongoing viral circulation despite vaccination. No correlation between HBV and HDV genotypes was observed for coinfected patients in Yakutia (r = - 0.016069332). HDV-2b circulates in Russia in Yakutia only and resulted from a single wave of introduction from Central Asia 135 years ago (95% HPD: 60-350 years), while HDV-1 strains resulted from multiple introductions from Europe, the Middle East, Central Asia, and different parts of Russia starting 180 years ago (95% HPD: 150-210 years) and continuing to the present day. The population dynamics of HDV-1 and HDV-2 show no signs of decline despite 20 years of HBV vaccination. The Birth-Death Skyline analysis showed an increase in the viral population in recent years for both HDV genotypes, indicating ongoing HDV epidemics. CONCLUSIONS: Taken together, these data call for strict control of HBV vaccination quality and coverage, and implementation of HBV and HDV screening programs in Yakutia.

Geographic and Temporal Variability of Hepatitis E Virus Circulation in the Russian Federation.

The factors influencing hepatitis E virus (HEV) circulation remain largely unexplored. We investigated HEV seroprevalence in humans and the prevalence of infection in farm pigs and rabbits in different regions of the Russian Federation, as well as the genetic diversity and population dynamics of the HEV. The anti-HEV IgG antibody detection rates in the general population increase significantly with age, from 1.5% in children and adolescents under 20 years old to 4.8% in adults aged between 20 and 59 years old to 16.7% in people aged 60 years and older. HEV seroprevalence varies between regions, with the highest rate observed in Belgorod Region (16.4% compared with the national average of 4.6%), which also has the country's highest pig population. When compared with the archival data, both increases and declines in HEV seroprevalence have been observed within the last 10 years, depending on the study region. Virus shedding has been detected in 19 out of the 21 pig farms surveyed. On one farm, the circulation of the same viral strain for five years was documented. All the human and animal strains belonged to the HEV-3 genotype, with its clade 2 sequences being predominant in pigs. The sequences are from patients, pigs, and sewage from pig farms clustered together, suggesting a zoonotic infection in humans and possible environmental contamination. The HEV-3 population size that was predicted using SkyGrid reconstruction demonstrated exponential growth in the 1970s-1990s, with a subsequent decline followed by a short rise around the year 2010, the pattern being similar to the dynamics of the pig population in the country. The HEV-3 reproduction number (Re) that was predicted using birth-death skyline analysis has fluctuated around 1 over the past 20 years in Russia but is 10 times higher in Belgorod Region. In conclusion, the HEV-3 circulation varies both geographically and temporally, even within a single country. The possible factors contributing to this variability are largely related to the circulation of the virus among farm pigs.

Coverage with Timely Administered Vaccination against Hepatitis B Virus and Its Influence on the Prevalence of HBV Infection in the Regions of Different Endemicity.

Universal hepatitis B vaccination of newborns was implemented in Russia starting from 1998. From 1998 to 2019, the incidence of acute hepatitis B reduced from 43.8 to 0.57 cases per 100,000 population. Here, we assessed the timely coverage of newborns with the birth dose (HepB-BD), second dose (HepB-2nd), and three vaccine doses (HepB3) in two remote regions of Russia with low (Belgorod Oblast) and high (Yakutia) levels of hepatitis B virus (HBV) endemicity. Vaccination data were obtained from the medical records of 1000 children in Yakutia and 2182 children in Belgorod Oblast. Sera of healthy volunteers from Belgorod Oblast (n = 1754) and Yakutia (n = 1072) across all age groups were tested for serological markers of HBV to assess the infection prevalence and herd immunity. Average HepB-BD coverage was 99.2% in Yakutia and 89.4% in Belgorod Oblast (p < 0.0001) and in both regions varied significantly, from 66% to 100%, between medical centers. The principal reason for the absence of HepB-BD was parent refusal, which accounted for 63.5% of cases of non-vaccination (83/123). While timely HepB-2nd coverage was only 55.4%-64.7%: HepB3 coverage by the age of one year exceeded 90% in both study regions. HBV surface antigen (HBsAg) prevalence in the 1998-2019 birth cohort was 0.2% (95% CI: 0.01-1.3%) in Belgorod Oblast and 3.2% (95% CI: 1.9-5.2%) in Yakutia. The proportion of persons testing negative for both antibodies to HBsAg (anti-HBs) and antibodies to HBV core antigen (anti-HBc) in the 1998-2019 birth cohort was 26.2% (125/481) in Belgorod Oblast and 32.3% (162/501) in Yakutia. We also assessed the knowledge of and attitude towards vaccination among 782 students and teachers of both medical and non-medical specialties from Belgorod State University. Only 60% of medical students knew that hepatitis B is a vaccine-preventable disease. Both medical and nonmedical students, 37.8% and 31.3%, respectively, expressed concerns about safety and actual necessity of vaccination. These data indicate the need to introduce a vaccine delivery audit system, improve medical education with respect to vaccination strategies and policies, and reinforce public knowledge on the benefits of vaccination.

Factors Influencing the Prevalence of Resistance-Associated Substitutions in NS5A Protein in Treatment-Naive Patients with Chronic Hepatitis C.

Direct-acting antivirals (DAAs) revolutionized treatment of hepatitis C virus (HCV) infection. Resistance-associated substitutions (RASs) present at the baseline impair response to DAA due to rapid selection of resistant HCV strains. NS5A is indispensable target of the current DAA treatment regimens. We evaluated prevalence of RASs in NS5A in DAA-naïve patients infected with HCV 1a (n = 19), 1b (n = 93), and 3a (n = 90) before systematic DAA application in the territory of the Russian Federation. Total proportion of strains carrying at least one RAS constituted 35.1% (71/202). In HCV 1a we detected only M28V (57.9%) attributed to a founder effect. Common RASs in HCV 1b were R30Q (7.5%), L31M (5.4%), P58S (4.4%), and Y93H (5.4%); in HCV 3a, A30S (31.0%), A30K (5.7%), S62L (8.9%), and Y93H (2.2%). Prevalence of RASs in NS5A of HCV 1b and 3a was similar to that worldwide, including countries practicing massive DAA application, i.e., it was not related to treatment. NS5A with and without RASs exhibited different co-variance networks, which could be attributed to the necessity to preserve viral fitness. Majority of RASs were localized in polymorphic regions subjected to immune pressure, with selected substitutions allowing immune escape. Altogether, this explains high prevalence of RAS in NS5A and low barrier for their appearance in DAA-inexperienced population.

Universal Single-Dose Vaccination against Hepatitis A in Children in a Region of High Endemicity.

Since August 2012, universal single-dose vaccination in children aged at least three years has been implemented in the Republic of Tuva, which was previously the region most affected by hepatitis A in Russia. The objective of this cross-sectional study was the assessment of the immunological and epidemiological effectiveness of vaccination program five years following its implementation. In the pre-vaccination period, anti-HAV antibody detection rates in Tuva was 66.0% [95% CI: 56.3-74.6%] in children aged 10-14 years and reached a plateau (>95%) by age 20-29 years. Annual incidence rates in children under 18 years of age peaked at 450-860 per 100,000 in pre-vaccination years but dropped to 7.5 per 100,000 in this age group and to 3.2 per 100,000 in the total population one year after the start of vaccination. Since 2016, no cases of hepatitis A has been reported in Tuva. Serum anti-HAV antibodies were quantified in samples from healthy children following single-dose vaccination. Protective anti-HAV antibody concentrations (≥10 mIU/mL) were detected in 98.0% (95% CI: 96.2-99.0% (442/451)) of children tested one month after single-dose immunization, in 93.5% (95% CI: 91.0-95.4% (477/510)) and in 91.1% (95% CI: 88.2-93.4% (422/463)) of children one year and five years after single-dose immunization, respectively. Anti-HAV antibody geometric mean concentrations were similar in sera collected one month, one year, and five years following single-dose vaccination: 40.24 mIU/mL, 44.96 mIU/mL, and 57.73 mIU/mL, respectively (p > 0.05). These data confirm that single-dose vaccination is an effective method of bringing hepatitis A under control in a short period of time in a highly endemic region.

Community screening for hepatitis C virus infection in a low-prevalence population.

BACKGROUND: Age cohort screening for hepatitis C virus (HCV) might be an effective strategy if the majority of undiagnosed cases are concentrated in a particular age group. The objective of this study was to determine HCV prevalence in different age cohorts of the general population in the Central European part of Russia and second, to assess feasibility of HCV antigen testing for community screening programs. METHODS: Sera from 2027 volunteers were tested for anti-HCV (Architect Anti-HCV, Abbott Laboratories). All anti-HCV reactive samples were confirmed in an immunoblot and tested for HCV Ag (ARCHITECT HCV Ag, Abbott Laboratories), HCV RNA and HCV viral load. RESULTS: Out of 31 individuals with anti-HCV reactive result, 22 (71%) were confirmed by immunoblot, six were false positives and three were indeterminate. Active infection was observed in 73% of anti-HCV confirmed positives. Five out of 16 individuals had low HCV-RNA levels (< 10,000 IU/mL) and one of those had a very low level (594 IU/mL). Agreement between HCV Ag and HCV RNA was 100%. Total anti-HCV and active HCV infection rates were 1.09% (22/2027) and 0.79% (16/2027), respectively. The peak rates were observed in people 60 years or older (anti-HCV: 2.84% [95% CI: 1.66-4.74%], 13/319; HCV RNA/HCV Ag: 2.23% [95% CI: 1.20-4.00%], 10/319). CONCLUSIONS: Overall HCV prevalence is low, except in people 60 years or older. The latter should be considered as a target group for HCV screening. The high agreement between HCV RNA and HCV Ag suggests the utility of HCV Ag testing to confirm active infection in screening programs.

Hepatitis C virus subtype 3a was introduced in the USSR in the early 1980s.

A total of 2120 nucleotide sequences of the NS5b region of HCV subtype 3a were analysed, including 310 strains derived from former republics of the USSR (Azerbaijan, Estonia, Lithuania, Russia, Tajikistan and Uzbekistan). Among the viral isolates collected from former regions of the Soviet Union, 294 strains formed 3 sustained phylogenetic clusters, with each having a common origin. Phylodynamic analysis demonstrated that the most recent common ancestors of the current strains inside the three clusters were introduced into the USSR population in 1981±1, 1984±2 and 1985±2, respectively (the confidence intervals were calculated using Student's t-distribution, P<0.05). The time estimation obtained for HCV subtype 3a correlated well with the historical and epidemiological context of this period, and in particular with the start of widespread injection drug use in the USSR in the first half of the 1980s.

Impact of Universal Hepatitis B Vaccination on Prevalence, Infection-Associated Morbidity and Mortality, and Circulation of Immune Escape Variants in Russia.

METHODS: 6,217 sera samples collected from volunteers in six epidemiologically different regions of Russia were tested for serological and molecular markers of HBV infection. A mathematical model developed by the U.S. Centers for Disease Control and Prevention was used to estimate the effect of vaccination and birth dose coverage on the incidence of HB and adverse outcomes of infection. RESULTS: Prevalence of HBsAg in the study population varied from 1.2% to 8.2%; anti-HBc detection rates were 13.0-46.2%. HBsAg detection rates in epidemiologically significant cohorts were 0.6-10.5% in women of childbearing age; 0-2.4% in children ≤5 years old; 1.9-8.1% in adults ≥30 years old. Mathematical modeling demonstrated that the current 96.1-99.6% level of birth dose coverage increased the effectiveness of vaccination 10-21 times compared to 50% and 0% birth dose coverage scenarios. HBV DNA was detected in 63 sera samples. The frequency of amino acid substitutions in HBsAg was 38% (24/63). Only in 3% (2/63) the mutations were within the a-determinant of HBsAg (M133T and G145S, one case each). None of the identified mutations eluded HBsAg detection, since all these samples tested positive for HBsAg by commercial ELISA. CONCLUSION: Despite a significant decline in acute HB incidence after the introduction of universal vaccination, many undiagnosed potential sources of infection remain. Low prevalence of HBV immune escape variants is a favorable predictor of vaccine effectiveness in the future.

Development of pan-phlebovirus RT-PCR assay.

This study reports the pan-phlebovirus assay capable of detecting both sandfly/mosquito- and tick-borne phleboviruses. Sensitivity and specificity of the assay was verified using a panel of arboviruses. The RT-PCR assay is simple and sensitive, and thus well suited for screening of field samples.

Mutational robustness and resilience of a replicative cis-element of RNA virus: Promiscuity, limitations, relevance.

Since replication of RNA-viruses is generally a low-fidelity process, it would be advantageous, if specific interactions of their genomic cis-elements with dedicated ligands are relatively tolerant to mutations. The specificity/promiscuity trade-off of such interactions was addressed here by investigating structural requirements of the oriL (also known as the clover leaf-like element), of poliovirus RNA, a replicative cis-element containing a conserved essential tetraloop functionally interacting with the viral protein 3CD. The sequence of this tetraloop and 2 adjacent base-pairs was randomized in the viral genome, and viable viruses were selected in susceptible cells. Strikingly, each position of this octanucleotide in 62 investigated viable viruses could be occupied by any nucleotide (with the exception of one position, which lacked U), though with certain sequence preferences, confirmed by engineering mutant viral genomes whose phenotypic properties were found to correlate with the strength of the cis-element/ligand interaction. The results were compatible with a hypothesis that functional recognition by 3CD requires that this tetraloop should stably or temporarily adopt a YNMG-like (Y=U/C, N=any nucleotide, M=A/C) fold. The fitness of "weak" viruses could be increased by compensatory mutations "improving" the tetraloops. Otherwise, the recognition of "bad" tetraloops might be facilitated by alterations in the 3CD protein. The virus appeared to tolerate mutations in its cis-element relaying on either robustness (spatial structure degeneracy) or resilience (a combination of dynamic RNA folding, low-fidelity replication modifying the cis-element or its ligand, and negative selection). These mechanisms (especially resilience involving metastable low-fit intermediates) can also contribute to the viral evolvability.

Recombination in hepatitis A virus: evidence for reproductive isolation of genotypes.

This study analysed phylogenetic evidence of recombination in sequences of hepatitis A virus (HAV) available in international databases. Isolation of distinct recombinant HAV strains has been reported previously; however, the prevalence of natural recombination and its role in HAV genetics remains obscure. Analysis of full genome sequences revealed evidence of common intratypic recombination among the most prevalent subtypes, IA and IIIA. Many of the available complete sequences of these genotypes carried phylogenetic signs of recombination in all genomic regions without obvious hotspots. In addition, and in line with previous reports, recombination between subtypes IA and IB was detected. A dataset of 104 published HAV sequences for the VP1-2A and 3CD genomic regions was also analysed. Multiple instances of phylogenetic incompatibility were found among subtypes IA and IIIA. Three cases of recombination disrupted the phylogenetic grouping of subtype IA HAV strains isolated in Japan within less than 4 years, indicating common intratypic recombination in HAV. There were no signs of recombination between different HAV genotypes, despite the fact that co-circulation of genotypes IA and IIIA has commonly been reported in different parts of the world and many sequences in the sampling in this study originated from the same geographical region. These results indicate that there is reproductive isolation between genotypes of HAV, as exists between enterovirus species, and suggest that common intratypic recombination constrains the diversity within a genotype and maintains HAV genotypes as global gene pools.

Hepatitis C virus recombinants are rare even among intravenous drug users.

Systematic studies of the circulation of hepatitis C virus (HCV) recombinants in different parts of the world have been initiated only recently, and no detailed information on this subject is available. The aim of the current investigation was to determine the frequency of HCV recombinants in intravenous drug users (IVDU) from two European countries. HCV RNA from serum samples was tested by RT-PCR with primers derived from the core and NS5B regions with subsequent sequencing and genotype assignment. The 118 samples from Germany (100%) and 45 out of 47 (96%) sera from Russia demonstrated concordant genotyping results. In the two genotype discrepant sera from Russia 2k/1b recombinants were identified. In order to test the hypothesis that the individuals from the IVDU group might be multiply exposed to various genotypes, 145 out of 165 genotyped serum samples, which were found to be positive for anti-NS4 antibodies, were serotyped with the Murex HCV serotyping kit that is based on detection of antibodies to type-specific peptides derived from the NS4 proteins of different HCV genotypes. Discrepancy in genotype and serotype attributions was observed in 11% cases. Retesting of 99 type 1a or 3a samples with a set of type- and subtype-specific primers revealed the presence of a mixed infection only in one case (1a/3a). Thus, the cases of the mixed infection with different HCV genotypes as well as the recombinant forms of HCV are very rare even in such a highly exposed group as IVDU.

Acute GB virus B infection of marmosets is accompanied by mutations in the NS5A protein.

GBV-B, a member of the Flaviviridae family of viruses, is the virus most closely related to HCV, and GBV-B infection in tamarin monkeys might represent a valuable surrogate animal model of HCV infection. In the current study, GBV-B was successfully transmitted to two marmosets (Callithrix jaccus). The infection resulted in viremia of 14- and 17-week duration, respectively, and was accompanied by elevation of isocitrate dehydrogenase activity. These data confirm that marmosets might represent an attractive model for GBV-B infection. The sequence of GBV-B NS5A, which was previously reported to have one of the highest mutation rates during infection in tamarins, was determined for viruses recovered from the inoculum and from marmoset blood samples obtained at weeks 1, 8, and 14 post inoculation in one marmoset and at weeks 2, 8, and 17 post inoculation in the other marmoset. In both animals, we detected four substitutions (R1945K, K2052G, F2196L, and G2268E), in the virus recovered immediately before viral clearance. Interestingly, two of these mutations (F2196L and G2268E) were described recently for viruses recovered from persistently infected tamarins. Appearance of these mutations presumably reflects a mechanism of immune escape rather than adaptation of the virus to a new host.