Serena A. Carroll

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.

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.

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.

Genetic Evidence for Rift Valley Fever Outbreaks in Madagascar Resulting from Virus Introductions from the East African Mainland rather than Enzootic Maintenance

ABSTRACT Rift Valley fever virus (RVFV), a mosquito-borne phlebovirus, has been detected in Madagascar since 1979, with occasional outbreaks. In 2008 to 2009, a large RVFV outbreak was detected in Malagasy livestock and humans during two successive rainy seasons. To determine whether cases were due to enzootic maintenance of the virus within Madagascar or to importation from the East African mainland, nine RVFV whole genomic sequences were generated for viruses from the 1991 and 2008 Malagasy outbreaks. Bayesian coalescent analyses of available whole S, M, and L segment sequences were used to estimate the time to the most recent common ancestor for the RVFVs. The 1979 Madagascar isolate shared a common ancestor with strains on the mainland around 1972. The 1991 Madagascar isolates were in a clade distinct from that of the 1979 isolate and shared a common ancestor around 1987. Finally, the 2008 Madagascar viruses were embedded within a large clade of RVFVs from the 2006–2007 outbreak in East Africa and shared a common ancestor around 2003 to 2004. These results suggest that the most recent Madagascar outbreak was caused by a virus likely arriving in the country some time between 2003 and 2008 and that this outbreak may be an extension of the 2006–2007 East African outbreak. Clustering of the Malagasy sequences into subclades indicates that the viruses have continued to evolve during their short-term circulation within the country. These data are consistent with the notion that RVFV outbreaks in Madagascar result not from emergence from enzootic cycles within the country but from recurrent virus introductions from the East African mainland.

Ancient common ancestry of Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne RNA virus responsible for outbreaks of severe hemorrhagic fever in humans. Although CCHF was first detected in the 1940s, high levels of genomic diversity argue against a recent origin. Here, Bayesian coalescent analyses were used to estimate the rate of evolution and relative age of the virus. A total of 43 S, 34 M, and 23 L segment sequences from samples collected between 1956 and 2005 were analyzed from across the broad geographic range of the virus. Using a relaxed molecular clock model, nucleotide substitutions were estimated to have occurred at a rate of 1.09x10(-4), 1.52x10(-4), and 0.58x10(-4) substitutions/site/year for the S, M, and L segments, respectively. The most recent common ancestor of the viruses existed approximately 3100-3500 years before present, or around 1500-1100 BC. Changes in agricultural practices and climate occurring near the time of the most recent common ancestor of CCHFV may have contributed to its emergence and spread.

Recent Ancestry of Kyasanur Forest Disease Virus

Clinicians in Asia should consider this disease when diagnosing acute febrile illnesses.

Ancient Ancestry of KFDV and AHFV Revealed by Complete Genome Analyses of Viruses Isolated from Ticks and Mammalian Hosts

Background Alkhurma hemorrhagic fever virus (AHFV) and Kyasanur forest disease virus (KFDV) cause significant human disease and mortality in Saudi Arabia and India, respectively. Despite their distinct geographic ranges, AHFV and KFDV share a remarkably high sequence identity. Given its emergence decades after KFDV, AHFV has since been considered a variant of KFDV and thought to have arisen from an introduction of KFDV to Saudi Arabia from India. To gain a better understanding of the evolutionary history of AHFV and KFDV, we analyzed the full length genomes of 16 AHFV and 3 KFDV isolates. Methodology/Principal Findings Viral genomes were sequenced and compared to two AHFV sequences available in GenBank. Sequence analyses revealed higher genetic diversity within AHFVs isolated from ticks than human AHFV isolates. A Bayesian coalescent phylogenetic analysis demonstrated an ancient divergence of AHFV and KFDV of approximately 700 years ago. Conclusions/Significance The high sequence diversity within tick populations and the presence of competent tick vectors in the surrounding regions, coupled with the recent identification of AHFV in Egypt, indicate possible viral range expansion or a larger geographic range than previously thought. The divergence of AHFV from KFDV nearly 700 years ago suggests other AHFV/KFDV-like viruses might exist in the regions between Saudi Arabia and India. Given the human morbidity and mortality associated with these viruses, these results emphasize the importance of more focused study of these significant public health threats.

High Diversity and Ancient Common Ancestry of Lymphocytic Choriomeningitis Virus

The diversity of these viruses has practical implications for the design of molecular diagnostic assays.

Rift Valley Fever, Sudan, 2007 and 2010

Viral sequences analyzed indicate recent virus movement and support the need for surveillance.

Ecology of Catarina Virus (family Arenaviridae) in Southern Texas, 2001–2004

A total of 3941 rodents were captured during a 46-month prospective (mark-recapture) study on the ecology of Catarina virus in southern Texas. Antibody reactive against Catarina virus was found in 73 (11.9%) of 611 southern plains woodrats (Neotoma micropus) and none of 3330 other rodents; strains of Catarina virus were isolated from 6 antibody-negative and 9 antibody-positive southern plains woodrats; and the infections in at least 3 southern plains woodrats were chronic. These results affirm the notion that the southern plains woodrat is the principal host of Catarina virus and suggest that Catarina virus infection is highly specific to N. micropus.