Marina L. Khristova

Genetic Detection and Characterization of Lujo Virus, a New Hemorrhagic Fever–Associated Arenavirus from Southern Africa

Lujo virus (LUJV), a new member of the family Arenaviridae and the first hemorrhagic fever–associated arenavirus from the Old World discovered in three decades, was isolated in South Africa during an outbreak of human disease characterized by nosocomial transmission and an unprecedented high case fatality rate of 80% (4/5 cases). Unbiased pyrosequencing of RNA extracts from serum and tissues of outbreak victims enabled identification and detailed phylogenetic characterization within 72 hours of sample receipt. Full genome analyses of LUJV showed it to be unique and branching off the ancestral node of the Old World arenaviruses. The virus G1 glycoprotein sequence was highly diverse and almost equidistant from that of other Old World and New World arenaviruses, consistent with a potential distinctive receptor tropism. LUJV is a novel, genetically distinct, highly pathogenic arenavirus.

Isolation of Genetically Diverse Marburg Viruses from Egyptian Fruit Bats

In July and September 2007, miners working in Kitaka Cave, Uganda, were diagnosed with Marburg hemorrhagic fever. The likely source of infection in the cave was Egyptian fruit bats (Rousettus aegyptiacus) based on detection of Marburg virus RNA in 31/611 (5.1%) bats, virus-specific antibody in bat sera, and isolation of genetically diverse virus from bat tissues. The virus isolates were collected nine months apart, demonstrating long-term virus circulation. The bat colony was estimated to be over 100,000 animals using mark and re-capture methods, predicting the presence of over 5,000 virus-infected bats. The genetically diverse virus genome sequences from bats and miners closely matched. These data indicate common Egyptian fruit bats can represent a major natural reservoir and source of Marburg virus with potential for spillover into humans.

Antibody Levels and Protection after Hepatitis B Vaccination: Results of a 15-Year Follow-up

Context Although administration of hepatitis B vaccine for infants is routine practice in many countries, we do not know whether the protection that this vaccine offers lasts beyond 10 years. Such information is essential to develop policies about booster vaccination. Contribution Of 841 Alaska Natives who received 3 doses of hepatitis B vaccination during 19811982 and were followed for 15 years, 84% had protective levels of antibody to hepatitis B surface antigen that indicated continued protection. The greatest decline in antibody levels occurred in people who received vaccine before 4 years of age. Definite asymptomatic breakthrough infections occurred in 16 participants. Cautions Only about half of the initial cohort of 1578 was available for testing at 15 years. The Editors Universal vaccination of infants with hepatitis B vaccine is included in the immunization programs of most countries and has been shown to be effective in reducing the rate of chronic hepatitis B virus (HBV) infection (1, 2). Protection has been demonstrated in persons and populations vaccinated for 5 to 10 years, and rates of asymptomatic breakthrough HBV infection have been extremely low (3-9). However, the duration of protection afforded by hepatitis B vaccination beyond 10 years and the possible need for booster doses of this vaccine are unknown. Alaska Natives have a high prevalence of chronic HBV infection, primarily acquired during early childhood (10). Between November 1981 and May 1982, Alaska Natives residing in 15 villages in southwest Alaska were enrolled in a cohort study to ascertain the immunogenicity and long-term effectiveness of hepatitis B vaccination (11-14). We report data on the persistence of antibodies to hepatitis B surface antigen (anti-HBs), incidence of HBV infection, and the genetic characteristics of the HBV isolates in persons with breakthrough infections 15 years after initial vaccination of this cohort. Methods Participants and Data Collection A total of 1578 Alaska Natives who were serologically negative for hepatitis B surface antigen (HBsAg) and antibody to hepatitis B core antigen (anti-HBc) were vaccinated on a 0-, 1-, and 6-month schedule with 3 doses of plasma-derived hepatitis B vaccine (Heptavax, Merck & Co., Inc., West Point, Pennsylvania) beginning in 1981 (11). Persons younger than 20 years of age received the 10-g dose, and adults received the 20-g dose. Of the 1578 persons vaccinated, 1436 (91%) were tested for an anti-HBs response 6 months after the last vaccine dose. From 1982 to 1992, serum specimens were obtained annually and once again during 1996 from as many of the 1578 consenting participants as possible. The Institutional Review Boards of the Alaska Area Native Health Service, the Indian Health Service, the Centers for Disease Control and Prevention, and the Yukon-Kuskokwim Health Corporation and the Norton Sound Regional Alaska Native health boards approved this study. All participants 18 years of age and older and parents of children younger than 18 years of age had provided signed informed consent to participate in the study; children older than 7 years of age gave verbal assent. The number of HBsAg-positive persons in each village was obtained from a registry used to follow patients with chronic HBV infection (15). Serologic Testing All serum specimens were tested for HBsAg, anti-HBs, and anti-HBc by radioimmunoassay using commercial test kits (Abbott Laboratories, Abbott Park, Illinois). At the initial testing of the cohort for anti-HBs, results were reported in sample ratio units. However, subsequent anti-HBs results were reported in milli international units (mIU) per mL using a World Health Organization reference standard (12-14). To ensure comparability of results over time, all serum specimens from each participant with sufficient volume (99.9% of all specimens collected during the study) were retested to determine anti-HBs levels in mIU/mL. Detection of HBV DNA and Nucleic Acid Sequencing Hepatitis B virus DNA was extracted from serum specimens (50 L) of participants with serologic markers of HBV infection by using commercially available reagents (MasterPure Complete DNA and RNA Purification Kit, Epicentre Technologies, Madison, Wisconsin), as described previously (16, 17). The HBsAg genomic region was then amplified by dilution cloning polymerase chain reaction by using previously described primers and methods to identify circulating variants of HBsAg (16, 17). The polymerase chain reaction products were purified and the nucleic acid sequence of the amplified region were determined by using prism dye or dRhodamine terminator cycle reactions (Applied Biosystems, Foster City, California) and automated sequencing (ABI Model 373 or 377, Applied Biosystems) (18). Sequence data were further analyzed by Sequence Navigator (ABI) and GCG software (19). Definitions The initial anti-HBs level was measured 6 months after the third dose of vaccine and 1 year after the first dose of vaccine. Participants with an initial anti-HBs level of at least 10 mIU/mL were considered vaccine responders. An anti-HBs level of 2 mIU/mL or greater was considered a positive result on subsequent specimens. A booster response was defined as a 2-fold or greater increase in anti-HBs levels between serologic test results. A definite HBV infection in a participant was defined as 1) 2 or more consecutive serum specimens positive for anti-HBc more than 1 year after the initial vaccine dose, 2) a single positive anti-HBc result with a positive HBV DNA result, or 3) any HBsAg-positive test result. A possible HBV infection was defined as a single positive or 2 nonconsecutive positive anti-HBc results. Participants who developed anti-HBc were interviewed for history of icterus or other clinical signs or symptoms compatible with acute hepatitis, and village and hospital medical records were reviewed for evidence of an illness compatible with viral hepatitis. Statistical Analysis Among persons who inadvertently received additional doses of hepatitis B vaccine during the follow-up period, anti-HBs results after the additional vaccine dose or doses were excluded from the analyses. Results for anti-HBs among persons with definite HBV infections were excluded from analyses after anti-HBc appeared. The primary outcomes in this study were the cumulative number of persons with a definite HBV infection during all follow-up years and the anti-HBs levels at the 15-year follow-up. The definitions of age classes (0 to 4 years, 5 to 19 years, 20 to 49 years, 50 years) were similar to those in a previously published analysis of this cohort (14). Although these data have been presented previously (11-14), we have provided the proportion of persons initially responding to vaccination. Quantitative anti-HBs levels are presented as geometric mean concentrations (GMCs). In bivariate analyses, analysis of variance was used to test the 15-year anti-HBs concentrations (log-transformed). Incidence rates of definite HBV infection were compared by using the Fisher exact test. We analyzed factors associated with anti-HBs levels over the 15 years after the first vaccine dose by using a linear mixed model (PROC MIXED in SAS version 9.1, SAS Institute, Inc., Cary, North Carolina) (19). We chose a longitudinal mixed linear model because it makes inferences by using information from the entire cohort collected at all follow-up time points. Levels of anti-HBs were log-transformed before analysis, and concentrations of 0 mIU/mL were assigned a value of 1.0 mIU/mL. Factors considered in the model were time (entered as a continuous covariate; linear and quadratic term were considered), age class at initial vaccination, sex, the log of the initial anti-HBs level, presence of an HBsAg-infected person in the household at the start of the study, and the proportion of village residents with chronic HBV infection at the end of follow-up, along with interaction terms involving time. Significance of 1 factor alone, such as age or sex, is called a main effect and is not of primary interest for this presentation. Of primary interest are the interaction terms between time and other factors. A significant interaction of time with another variable, such as sex, indicates that the decline in anti-HBs level differed between males and females. We obtained parameter estimates by using restricted maximum likelihood. We used an unstructured covariance matrix to account for dependence of observations across time within individuals. Backward elimination of statistically nonsignificant terms yielded a final model of main effects and time interaction terms. If the time interaction term was statistically significant, the main effect term remained in the model regardless of statistical significance. We used the Wald chi-square statistic to test covariates. Contrast tests were 2-sided, and an level of 0.05 was required. We used residual plots to evaluate model fit. A secondary outcome was a boost in anti-HBs level at the 11- or 15-year follow-up among persons without additional doses of vaccine. We used the chi-square test or the CochranMantelHaenszel test to compare age and sex of persons with a booster response to those of persons without a booster response at both follow-up years. All P values were exact where appropriate and were 2-sided; results were considered statistically significant at the 0.05 level. We conducted analyses by using StatXact4 (Cytel Software Corp., Cambridge, Massachusetts) and SAS software, version 9.1 (SAS Institute, Inc.). Missing Data Throughout the entire study, anti-HBs determinations were observed for 68% of all potential observational time points (Appendix Table 1). The 15 remote rural Alaskan study villages are accessible only by airplane. During each year, study personnel flew into each village for 1 to 2 days and attempted to recruit all available participants. Persons not available or out of the village for the day were considered miss

Marburgvirus Genomics and Association with a Large Hemorrhagic Fever Outbreak in Angola

ABSTRACT In March 2005, the Centers for Disease Control and Prevention (CDC) investigated a large hemorrhagic fever (HF) outbreak in Uige Province in northern Angola, West Africa. In total, 15 initial specimens were sent to CDC, Atlanta, Ga., for testing for viruses associated with viral HFs known to be present in West Africa, including ebolavirus. Marburgvirus was also included despite the fact that the origins of all earlier outbreaks were linked directly to East Africa. Surprisingly, marburgvirus was confirmed (12 of 15 specimens) as the cause of the outbreak. The outbreak likely began in October 2004 and ended in July 2005, and it included 252 cases and 227 (90%) fatalities (report from the Ministry of Health, Republic of Angola, 2005), making it the largest Marburg HF outbreak on record. A real-time quantitative reverse transcription-PCR assay utilized and adapted during the outbreak proved to be highly sensitive and sufficiently robust for field use. Partial marburgvirus RNA sequence analysis revealed up to 21% nucleotide divergence among the previously characterized East African strains, with the most distinct being Ravn from Kenya (1987). The Angolan strain was less different (∼7%) from the main group of East African marburgviruses than one might expect given the large geographic separation. To more precisely analyze the virus genetic differences between outbreaks and among viruses within the Angola outbreak itself, a total of 16 complete virus genomes were determined, including those of the virus isolates Ravn (Kenya, 1987) and 05DRC, 07DRC, and 09DRC (Democratic Republic of Congo, 1998) and the reference Angolan virus isolate (Ang1379v). In addition, complete genome sequences were obtained from RNAs extracted from 10 clinical specimens reflecting various stages of the disease and locations within the Angolan outbreak. While the marburgviruses exhibit high overall genetic diversity (up to 22%), only 6.8% nucleotide difference was found between the West African Angolan viruses and the majority of East African viruses, suggesting that the virus reservoir species in these regions are not substantially distinct. Remarkably few nucleotide differences were found among the Angolan clinical specimens (0 to 0.07%), consistent with an outbreak scenario in which a single (or rare) introduction of virus from the reservoir species into the human population was followed by person-to-person transmission with little accumulation of mutations. This is in contrast to the 1998 to 2000 marburgvirus outbreak, where evidence of several virus genetic lineages (with up to 21% divergence) and multiple virus introductions into the human population was found.

Crimean-Congo Hemorrhagic Fever Virus Genomics and Global Diversity

ABSTRACT Crimean-Congo hemorrhagic fever (CCHF) is a severe illness with high case fatality that occurs in Africa, Europe, the Middle East, and Asia. The complete genomes of 13 geographically and temporally diverse virus strains were determined, and CCHF viruses were found to be highly variable with 20 and 8%, 31 and 27%, and 22 and 10% nucleotide and deduced amino acid differences detected among virus S (nucleocapsid), M (glycoprotein), and L (polymerase) genome segments, respectively. Distinct geographic lineages exist, but with multiple exceptions indicative of long-distance virus movement. Discrepancies among the virus S, M, and L phylogenetic tree topologies document multiple RNA segment reassortment events. An analysis of individual segment datasets suggests genetic recombination also occurs. For an arthropod-borne virus, the genomic plasticity of CCHF virus is surprisingly high.

Complete Genome Analysis of 33 Ecologically and Biologically Diverse Rift Valley Fever Virus Strains Reveals Widespread Virus Movement and Low Genetic Diversity due to Recent Common Ancestry

ABSTRACT Rift Valley fever (RVF) virus is a mosquito-borne RNA virus responsible for large explosive outbreaks of acute febrile disease in humans and livestock in Africa with significant mortality and economic impact. The successful high-throughput generation of the complete genome sequence was achieved for 33 diverse RVF virus strains collected from throughout Africa and Saudi Arabia from 1944 to 2000, including strains differing in pathogenicity in disease models. While several distinct virus genetic lineages were determined, which approximately correlate with geographic origin, multiple exceptions indicative of long-distance virus movement have been found. Virus strains isolated within an epidemic (e.g., Mauritania, 1987, or Egypt, 1977 to 1978) exhibit little diversity, while those in enzootic settings (e.g., 1970s Zimbabwe) can be highly diverse. In addition, the large Saudi Arabian RVF outbreak in 2000 appears to have involved virus introduction from East Africa, based on the close ancestral relationship of a 1998 East African virus. Virus genetic diversity was low (∼5%) and primarily involved accumulation of mutations at an average of 2.9 × 10−4 substitutions/site/year, although some evidence of RNA segment reassortment was found. Bayesian analysis of current RVF virus genetic diversity places the most recent common ancestor of these viruses in the late 1800s, the colonial period in Africa, a time of dramatic changes in agricultural practices and introduction of nonindigenous livestock breeds. In addition to insights into the evolution and ecology of RVF virus, these genomic data also provide a foundation for the design of molecular detection assays and prototype vaccines useful in combating this important disease.

Seasonal Pulses of Marburg Virus Circulation in Juvenile Rousettus aegyptiacus Bats Coincide with Periods of Increased Risk of Human Infection

Marburg virus (family Filoviridae) causes sporadic outbreaks of severe hemorrhagic disease in sub-Saharan Africa. Bats have been implicated as likely natural reservoir hosts based most recently on an investigation of cases among miners infected in 2007 at the Kitaka mine, Uganda, which contained a large population of Marburg virus-infected Rousettus aegyptiacus fruit bats. Described here is an ecologic investigation of Python Cave, Uganda, where an American and a Dutch tourist acquired Marburg virus infection in December 2007 and July 2008. More than 40,000 R. aegyptiacus were found in the cave and were the sole bat species present. Between August 2008 and November 2009, 1,622 bats were captured and tested for Marburg virus. Q-RT-PCR analysis of bat liver/spleen tissues indicated ∼2.5% of the bats were actively infected, seven of which yielded Marburg virus isolates. Moreover, Q-RT-PCR-positive lung, kidney, colon and reproductive tissues were found, consistent with potential for oral, urine, fecal or sexual transmission. The combined data for R. aegyptiacus tested from Python Cave and Kitaka mine indicate low level horizontal transmission throughout the year. However, Q-RT-PCR data show distinct pulses of virus infection in older juvenile bats (∼six months of age) that temporarily coincide with the peak twice-yearly birthing seasons. Retrospective analysis of historical human infections suspected to have been the result of discrete spillover events directly from nature found 83% (54/65) events occurred during these seasonal pulses in virus circulation, perhaps demonstrating periods of increased risk of human infection. The discovery of two tags at Python Cave from bats marked at Kitaka mine, together with the close genetic linkages evident between viruses detected in geographically distant locations, are consistent with R. aegyptiacus bats existing as a large meta-population with associated virus circulation over broad geographic ranges. These findings provide a basis for developing Marburg hemorrhagic fever risk reduction strategies.

Multiple Virus Lineages Sharing Recent Common Ancestry Were Associated with a Large Rift Valley Fever Outbreak among Livestock in Kenya during 2006-2007

ABSTRACT Rift Valley fever (RVF) virus historically has caused widespread and extensive outbreaks of severe human and livestock disease throughout Africa, Madagascar, and the Arabian Peninsula. Following unusually heavy rainfall during the late autumn of 2006, reports of human and animal illness consistent with RVF virus infection emerged across semiarid regions of the Garissa District of northeastern Kenya and southern Somalia. Following initial RVF virus laboratory confirmation, a high-throughput RVF diagnostic facility was established at the Kenyan Central Veterinary Laboratories in Kabete, Kenya, to support the real-time identification of infected livestock and to facilitate outbreak response and control activities. A total of 3,250 specimens from a variety of animal species, including domesticated livestock (cattle, sheep, goats, and camels) and wildlife collected from a total of 55 of 71 Kenyan administrative districts, were tested by molecular and serologic assays. Evidence of RVF infection was found in 9.2% of animals tested and across 23 districts of Kenya, reflecting the large number of affected livestock and the geographic extent of the outbreak. The complete S, M, and/or L genome segment sequence was obtained from a total of 31 RVF virus specimens spanning the entire known outbreak period (December-May) and geographic areas affected by RVF virus activity. Extensive genomic analyses demonstrated the concurrent circulation of multiple virus lineages, gene segment reassortment, and the common ancestry of the 2006/2007 outbreak viruses with those from the 1997-1998 east African RVF outbreak. Evidence of recent increases in genomic diversity and effective population size 2 to 4 years prior to the 2006-2007 outbreak also was found, indicating ongoing RVF virus activity and evolution during the interepizootic/epidemic period. These findings have implications for further studies of basic RVF virus ecology and the design of future surveillance/diagnostic activities, and they highlight the critical need for safe and effective vaccines and antiviral compounds to combat this significant veterinary and public health threat.

Persistence of antibody to hepatitis B and protection from disease among Alaska natives immunized at birth.

Background: Alaska Native (AN) children were at high risk of acquiring hepatitis B virus (HBV) infection before vaccination began in 1983. We evaluated the long-term protection from hepatitis B (HB) vaccination among AN children immunized when infants. Methods: During 1984–1995, we recruited a convenience sample of AN children who had received a three dose series of HB vaccine starting at birth and had serum antibody to hepatitis B (anti-HBs) concentrations of ≥10 mIU/mL at 7–26 months of age. We evaluated anti-HBs concentrations and the presence of anti-HBc in participants’ sera every other year up to age 16 years. Anti-HB core antigen (anti-HBc)-positive specimens were tested for hepatitis B surface antigen and for HBV DNA. Results: We followed 334 children for 3151 person-years (median, 10 years per child) with 1610 specimens collected. Anti-HBs concentrations dropped rapidly among all participants. Among children 2, 5 and 10 years of age, 37 of 79 (47%), 33 of 176 (19%) and 8 of 95 (8%), respectively, had anti-HBs concentrations of ≥10 mIU/mL. Receipt of recombinant vaccine was significantly associated with a more rapid antibody decline (P < 0.001). Six (1.8%) children acquired anti-HBc, 3 of whom had definite breakthrough infections (at least 2 consecutive anti-HBc-positive specimens or at least 1 anti-HBc-positive specimen and HBV DNA detection by PCR). None of these children had detectable hepatitis B surface antigen, and none had symptoms of hepatitis. Conclusions: Anti-HBs concentrations declined over time among AN infants successfully immunized with HB vaccine starting at birth. Transient anti-HBc appeared in a small percentage of children; however, none developed clinical signs of hepatitis or chronic HBV infection.

Molecular evolution of viruses of the family Filoviridae based on 97 whole genome sequences

ABSTRACT Viruses in the Ebolavirus and Marburgvirus genera (family Filoviridae) have been associated with large outbreaks of hemorrhagic fever in human and nonhuman primates. The first documented cases occurred in primates over 45 years ago, but the amount of virus genetic diversity detected within bat populations, which have recently been identified as potential reservoir hosts, suggests that the filoviruses are much older. Here, detailed Bayesian coalescent phylogenetic analyses are performed on 97 whole-genome sequences, 55 of which are newly reported, to comprehensively examine molecular evolutionary rates and estimate dates of common ancestry for viruses within the family Filoviridae. Molecular evolutionary rates for viruses belonging to different species range from 0.46 × 10−4 nucleotide substitutions/site/year for Sudan ebolavirus to 8.21 × 10−4 nucleotide substitutions/site/year for Reston ebolavirus. Most recent common ancestry can be traced back only within the last 50 years for Reston ebolavirus and Zaire ebolavirus species and suggests that viruses within these species may have undergone recent genetic bottlenecks. Viruses within Marburg marburgvirus and Sudan ebolavirus species can be traced back further and share most recent common ancestors approximately 700 and 850 years before the present, respectively. Examination of the whole family suggests that members of the Filoviridae, including the recently described Lloviu virus, shared a most recent common ancestor approximately 10,000 years ago. These data will be valuable for understanding the evolution of filoviruses in the context of natural history as new reservoir hosts are identified and, further, for determining mechanisms of emergence, pathogenicity, and the ongoing threat to public health.