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.

Cell culture and electron microscopy for identifying viruses in diseases of unknown cause.

During outbreaks of infectious diseases or in cases of severely ill patients, it is imperative to identify the causative agent. This report describes several events in which virus isolation and identification by electron microscopy were critical to initial recognition of the etiologic agent, which was further analyzed by additional laboratory diagnostic assays. Examples include severe acute respiratory syndrome coronavirus, and Nipah, lymphocytic choriomeningitis, West Nile, Cache Valley, and Heartland viruses. These cases illustrate the importance of the techniques of cell culture and electron microscopy in pathogen identification and recognition of emerging diseases.

Characterization of Nipah virus from outbreaks in Bangladesh, 2008-2010.

Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes fatal encephalitis in humans. The initial outbreak of NiV infection occurred in Malaysia and Singapore in 1998-1999; relatively small, sporadic outbreaks among humans have occurred in Bangladesh since 2001. We characterized the complete genomic sequences of identical NiV isolates from 2 patients in 2008 and partial genomic sequences of throat swab samples from 3 patients in 2010, all from Bangladesh. All sequences from patients in Bangladesh comprised a distinct genetic group. However, the detection of 3 genetically distinct sequences from patients in the districts of Faridpur and Gopalganj indicated multiple co-circulating lineages in a localized region over a short time (January-March 2010). Sequence comparisons between the open reading frames of all available NiV genes led us to propose a standardized protocol for genotyping NiV; this protcol provides a simple and accurate way to classify current and future NiV sequences.

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.

Sin Nombre virus-specific immunoglobulin M and G kinetics in hantavirus pulmonary syndrome and the role played by serologic responses in predicting disease outcome.

BACKGROUND: Sin Nombre virus (SNV) is the primary cause of hantavirus pulmonary syndrome (HPS) in the United States. Although other studies have demonstrated a possible association between neutralizing antibody titers and the severity of HPS, the exact nature of serologic responses and their association with outcomes have not been fully characterized. METHODS: We examined immunoglobulin M (IgM) and immunoglobulin G (IgG) serologic responses in 94 clinical samples from 81 patients with confirmed HPS. We further compared a subset of 31 patients with fatal HPS and 20 surviving patients for whom samples were available within a week after the onset of HPS. RESULTS: SNV-specific IgM antibodies displayed a trend suggesting an early peak, whereas IgG antibody values peaked later. Among individuals with samples from the first week after the onset of HPS, all surviving patients had SNV-specific IgG responses, compared with <50% of patients with fatal HPS, and the distribution of IgG responses was significantly higher in surviving patients. CONCLUSIONS: Production of SNV-specific IgM antibodies occurs early during the clinical course of HPS, whereas production of IgG antibodies may be more protracted. The presence and overall distribution of higher IgG antibody titers in surviving patients with HPS suggests that production of SNV-specific IgG may be a strong predictor of favorable outcomes.

High diversity and ancient common ancestry of lymphocytic choriomeningitis virus.

Lymphocytic choriomeningitis virus (LCMV) is the prototype of the family Arenaviridae. LCMV can be associated with severe disease in humans, and its global distribution reflects the broad dispersion of the primary rodent reservoir, the house mouse (Mus musculus). Recent interest in the natural history of the virus has been stimulated by increasing recognition of LCMV infections during pregnancy, and in clusters of LCMV-associated fatal illness among tissue transplant recipients. Despite its public health importance, little is known regarding the genetic diversity or distribution of virus variants. Genomic analysis of 29 LCMV strains collected from a variety of geographic and temporal sources showed these viruses to be highly diverse. Several distinct lineages exist, but there is little correlation with time or place of isolation. Bayesian analysis estimates the most recent common ancestor to be 1,000-5,000 years old, and this long history is consistent with complex phylogeographic relationships of the extant virus isolates.

Chapare virus, a newly discovered arenavirus isolated from a fatal hemorrhagic fever case in Bolivia.

A small focus of hemorrhagic fever (HF) cases occurred near Cochabamba, Bolivia, in December 2003 and January 2004. Specimens were available from only one fatal case, which had a clinical course that included fever, headache, arthralgia, myalgia, and vomiting with subsequent deterioration and multiple hemorrhagic signs. A non-cytopathic virus was isolated from two of the patient serum samples, and identified as an arenavirus by IFA staining with a rabbit polyvalent antiserum raised against South American arenaviruses known to be associated with HF (Guanarito, Machupo, and Sabiá). RT-PCR analysis and subsequent analysis of the complete virus S and L RNA segment sequences identified the virus as a member of the New World Clade B arenaviruses, which includes all the pathogenic South American arenaviruses. The virus was shown to be most closely related to Sabiá virus, but with 26% and 30% nucleotide difference in the S and L segments, and 26%, 28%, 15% and 22% amino acid differences for the L, Z, N, and GP proteins, respectively, indicating the virus represents a newly discovered arenavirus, for which we propose the name Chapare virus. In conclusion, two different arenaviruses, Machupo and Chapare, can be associated with severe HF cases in Bolivia.

Newly discovered ebola virus associated with hemorrhagic fever outbreak in Uganda.

Over the past 30 years, Zaire and Sudan ebolaviruses have been responsible for large hemorrhagic fever (HF) outbreaks with case fatalities ranging from 53% to 90%, while a third species, Côte d'Ivoire ebolavirus, caused a single non-fatal HF case. In November 2007, HF cases were reported in Bundibugyo District, Western Uganda. Laboratory investigation of the initial 29 suspect-case blood specimens by classic methods (antigen capture, IgM and IgG ELISA) and a recently developed random-primed pyrosequencing approach quickly identified this to be an Ebola HF outbreak associated with a newly discovered ebolavirus species (Bundibugyo ebolavirus) distantly related to the Côte d'Ivoire ebolavirus found in western Africa. Due to the sequence divergence of this new virus relative to all previously recognized ebolaviruses, these findings have important implications for design of future diagnostic assays to monitor Ebola HF disease in humans and animals, and ongoing efforts to develop effective antivirals and vaccines.

Human febrile illness caused by encephalomyocarditis virus infection, Peru.

Etiologic studies of acute febrile disease were conducted in sites across South America, including Cusco and Iquitos, Peru. Patients' clinical signs and symptoms were recorded, and acute- and convalescent-phase serum samples were obtained for serologic examination and virus isolation in Vero E6 and C6/36 cells. Virus isolated in Vero E6 cells was identified as encephalomyocarditis virus (EMCV) by electron microscopy and by subsequent molecular diagnostic testing of samples from 2 febrile patients with nausea, headache, and dyspnea. The virus was recovered from acute-phase serum samples from both case-patients and identified with cardiovirus-specific reverse transcription-PCR and sequencing. Serum samples from case-patient 1 showed cardiovirus antibody by immunoglobulin M ELISA (acute phase <8, convalescent phase >1,024) and by neutralization assay (acute phase <10, convalescent phase >1,280). Serum samples from case-patient 2 did not contain antibodies detectable by either assay. Detection of virus in serum strongly supports a role for EMCV in human infection and febrile illness.

Recurrent zoonotic transmission of Nipah virus into humans, Bangladesh, 2001-2007.

Human Nipah outbreaks recur in a specific region and time of year in Bangladesh. Fruit bats are the reservoir host for Nipah virus. We identified 23 introductions of Nipah virus into human populations in central and northwestern Bangladesh from 2001 through 2007. Ten introductions affected multiple persons (median 10). Illness onset occurred from December through May but not every year. We identified 122 cases of human Nipah infection. The mean age of case-patients was 27 years; 87 (71%) died. In 62 (51%) Nipah virus-infected patients, illness developed 5-15 days after close contact with another Nipah case-patient. Nine (7%) Nipah case-patients transmitted virus to others. Nipah case-patients who had difficulty breathing were more likely than those without respiratory difficulty to transmit Nipah (12% vs. 0%, p = 0.03). Although a small minority of infected patients transmit Nipah virus, more than half of identified cases result from person-to-person transmission. Interventions to prevent virus transmission from bats to humans and from person to person are needed.

Transmission of lymphocytic choriomeningitis virus by organ transplantation.

BACKGROUND: In December 2003 and April 2005, signs and symptoms suggestive of infection developed in two groups of recipients of solid-organ transplants. Each cluster was investigated because diagnostic evaluations were unrevealing, and in each a common donor was recognized. METHODS: We examined clinical specimens from the two donors and eight recipients, using viral culture, electron microscopy, serologic testing, molecular analysis, and histopathological examination with immunohistochemical staining to identify a cause. Epidemiologic investigations, including interviews, environmental assessments, and medical-record reviews, were performed to characterize clinical courses and to determine the cause of the illnesses. RESULTS: Laboratory testing revealed lymphocytic choriomeningitis virus (LCMV) in all the recipients, with a single, unique strain of LCMV identified in each cluster. In both investigations, LCMV could not be detected in the organ donor. In the 2005 cluster, the donor had had contact in her home with a pet hamster infected with an LCMV strain identical to that detected in the organ recipients; no source of LCMV infection was found in the 2003 cluster. The transplant recipients had abdominal pain, altered mental status, thrombocytopenia, elevated aminotransferase levels, coagulopathy, graft dysfunction, and either fever or leukocytosis within three weeks after transplantation. Diarrhea, peri-incisional rash, renal failure, and seizures were variably present. Seven of the eight recipients died, 9 to 76 days after transplantation. One recipient, who received ribavirin and reduced levels of immunosuppressive therapy, survived. CONCLUSIONS: We document two clusters of LCMV infection transmitted through organ transplantation.

Multiple virus lineages sharing recent common ancestry were associated with a Large Rift Valley fever outbreak among livestock in Kenya during 2006-2007.

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.

Clinical presentation of nipah virus infection in Bangladesh.

BACKGROUND: In Bangladesh, 4 outbreaks of Nipah virus infection were identified during the period 2001-2004. METHODS: We characterized the clinical features of Nipah virus-infected individuals affected by these outbreaks. We classified patients as having confirmed cases of Nipah virus infection if they had antibodies reactive with Nipah virus antigen. Patients were considered to have probable cases of Nipah virus infection if they had symptoms consistent with Nipah virus infection during the same time and in the same community as patients with confirmed cases. RESULTS: We identified 92 patients with Nipah virus infection, 67 (73%) of whom died. Although all age groups were affected, 2 outbreaks principally affected young persons (median age, 12 years); 62% of the affected persons were male. Fever, altered mental status, headache, cough, respiratory difficulty, vomiting, and convulsions were the most common signs and symptoms; clinical and radiographic features of acute respiratory distress syndrome of Nipah illness were identified during the fourth outbreak. Among those who died, death occurred a median of 6 days (range, 2-36 days) after the onset of illness. Patients who died were more likely than survivors to have a temperature >37.8 degrees C, altered mental status, difficulty breathing, and abnormal plantar reflexes. Among patients with Nipah virus infection who had well-defined exposure to another patient infected with Nipah virus, the median incubation period was 9 days (range, 6-11 days). CONCLUSIONS: Nipah virus infection produced rapidly progressive severe illness affecting the central nervous and respiratory systems. Clinical characteristics of Nipah virus infection in Bangladesh, including a severe respiratory component, appear distinct from clinical characteristics reported during earlier outbreaks in other countries.

A novel coronavirus associated with severe acute respiratory syndrome.

BACKGROUND: A worldwide outbreak of severe acute respiratory syndrome (SARS) has been associated with exposures originating from a single ill health care worker from Guangdong Province, China. We conducted studies to identify the etiologic agent of this outbreak. METHODS: We received clinical specimens from patients in seven countries and tested them, using virus-isolation techniques, electron-microscopical and histologic studies, and molecular and serologic assays, in an attempt to identify a wide range of potential pathogens. RESULTS: None of the previously described respiratory pathogens were consistently identified. However, a novel coronavirus was isolated from patients who met the case definition of SARS. Cytopathological features were noted in Vero E6 cells inoculated with a throat-swab specimen. Electron-microscopical examination revealed ultrastructural features characteristic of coronaviruses. Immunohistochemical and immunofluorescence staining revealed reactivity with group I coronavirus polyclonal antibodies. Consensus coronavirus primers designed to amplify a fragment of the polymerase gene by reverse transcription-polymerase chain reaction (RT-PCR) were used to obtain a sequence that clearly identified the isolate as a unique coronavirus only distantly related to previously sequenced coronaviruses. With specific diagnostic RT-PCR primers we identified several identical nucleotide sequences in 12 patients from several locations, a finding consistent with a point-source outbreak. Indirect fluorescence antibody tests and enzyme-linked immunosorbent assays made with the new isolate have been used to demonstrate a virus-specific serologic response. This virus may never before have circulated in the U.S. population. CONCLUSIONS: A novel coronavirus is associated with this outbreak, and the evidence indicates that this virus has an etiologic role in SARS. Because of the death of Dr. Carlo Urbani, we propose that our first isolate be named the Urbani strain of SARS-associated coronavirus.

Risk of nosocomial transmission of Nipah virus in a Bangladesh hospital.

We conducted a seroprevalence study and exposure survey of healthcare workers to assess the risk of nosocomial transmission of Nipah virus during an outbreak in Bangladesh in 2004. No evidence of recent Nipah virus infection was detected despite substantial exposures and minimal use of personal protective equipment.

Two cases of hantavirus pulmonary syndrome in Randolph County, West Virginia: a coincidence of time and place?

Hantavirus pulmonary syndrome (HPS) is caused by an infection with viruses of the genus Hantavirus in the western hemisphere. Rodent hosts of hantaviruses are present throughout the United States. In July 2004, two HPS case-patients were identified in Randolph County, WV: a wildlife science graduate student working locally and a Randolph County resident. We interviewed family members and colleagues, reviewed medical records, and conducted environmental studies at likely exposure sites. Small mammals were trapped, and blood, urine, and tissue samples were submitted to the Centers for Disease Control and Prevention for laboratory analyses. These analyses confirmed that both patients were infected with Monongahela virus, a Sin Nombre hantavirus variant hosted by the Cloudland deer mouse, Peromyscus maniculatus nubiterrae. Other than one retrospectively diagnosed case in 1981, these are the first HPS cases reported in West Virginia. These cases emphasize the need to educate the public throughout the United States regarding risks and prevention measures for hantavirus infection.

Serologic evidence of a Rickettsia akari-like infection among wild-caught rodents in Orange County and humans in Los Angeles County, California.

We detected antibodies reactive with Rickettsia akari, the etiologic agent of rickettsialpox in humans and in 83 of 359 (23%) rodents belonging to several species, collected in Orange County, CA. Reciprocal antibody titers >1:16 to R. akari were detected in native mice and rats (Peromyscus maniculatus, P. eremicus, and Neotoma fuscipes) and in Old World mice and rats (Mus musculus, Rattus rattus, and R. norvegicus), representing the first time that antibodies reactive with this agent have been detected in four of these species and the first report of these antibodies in rodents and humans west of the Mississippi River. We then tested serum samples from individuals who used a free clinic in downtown Los Angeles and found that 25 of 299 (8%) of these individuals had antibody titers >1:64 to R. akari. Serologic evidence suggested that R. akari or a closely related rickettsia is prevalent among several rodent species at these localities and that infection spills over into certain segments of the human population. Isolation or molecular confirmation of the agent is needed to conclusively state that R. akari is the etiologic agent infecting these rodents.

Congenital lymphocytic choriomeningitis virus: an underdiagnosed cause of neonatal hydrocephalus.

We report a case of congenital hydrocephalus caused by lymphocytic choriomeningitis virus with severe neurologic sequelae, including hydrocephalus, chorioretinitis, blindness and developmental delay. This is the first report of lymphocytic choriomeningitis virus isolation in the cerebrospinal fluid of a congenitally infected infant.

Monoclonal antibodies to SARS-associated coronavirus (SARS-CoV): identification of neutralizing and antibodies reactive to S, N, M and E viral proteins.

Monoclonal antibodies (Mabs) against the Urbani strain of the SARS-associated coronavirus (SARS-CoV) were developed and characterized for reactivity to SARS-CoV and SARS-CoV S, N, M, and E proteins using enzyme-linked immunoabsorbent (ELISA), radioimmunoprecipitation, immunofluorescence, Western Blot and microneutralization assays. Twenty-six mAbs were reactive to SARS-CoV by ELISA, and nine were chosen for detailed characterization. Five mAbs reacted against the S protein, two against the M protein, and one each against the N and E proteins. Two of five S protein mAbs neutralized SARS-CoV infection of Vero E6 cells and reacted to an epitope within amino acids 490-510 in the S protein. While two of the three non-neutralizing antibodies recognized at second epitope within amino acids 270-350. The mAbs characterized should prove useful for developing SARS-CoV diagnostic assays and for studying the biology of infection and pathogenesis of disease.

Rickettsia parkeri: a newly recognized cause of spotted fever rickettsiosis in the United States.

Ticks, including many that bite humans, are hosts to several obligate intracellular bacteria in the spotted fever group (SFG) of the genus Rickettsia. Only Rickettsia rickettsii, the agent of Rocky Mountain spotted fever, has been definitively associated with disease in humans in the United States. Herein we describe disease in a human caused by Rickettsia parkeri, an SFG rickettsia first identified >60 years ago in Gulf Coast ticks (Amblyomma maculatum) collected from the southern United States. Confirmation of the infection was accomplished using serological testing, immunohistochemical staining, cell culture isolation, and molecular methods. Application of specific laboratory assays to clinical specimens obtained from patients with febrile, eschar-associated illnesses following a tick bite may identify additional cases of R. parkeri rickettsiosis and possibly other novel SFG rickettsioses in the United States.