Emerging infections: lessons from the viral hemorrhagic fevers.

Two Institute of Medicine reports since 1992 have emphasized the dangerous and continuing threat to the world from emerging infectious diseases. Working with viral hemorrhagic fevers provides a number of lessons related to the processes that control emergence, the pattern of disease after emergence, and how to cope with these incidents. This short paper uses two arenavirus hemorrhagic fevers to illustrate some of these principles. Argentine and Bolivian hemorrhagic fevers first came to medical attention in the 1950's. The forces that underlie the emergence of disease in Argentina are not understood, but the Bolivian episode has a reasonably understandable train of events behind it. The Argentine disease had serious impact on the large agricultural economy, and the ecology of the rodent reservoir did not lend itself to control; a vaccine was developed by Argentina and the U.S. with the latter motivated largely by biodefense. The Bolivian disease was controlled in large part by eliminating rodents that invaded towns, and the impact was subsequently below the level needed to trigger drug or vaccine development. These two viruses were important in the recognition of a new family of viruses (Arenaviridae), and this finding of new taxons during the investigation of emerging infectious diseases continues.

Community ecology of small mammal populations in Panamá following an outbreak of Hantavirus pulmonary syndrome.

In late 1999 and early 2000, an outbreak of hantavirus pulmonary syndrome (HPS) occurred in and around Los Santos, on the Azuero Peninsula of southwestern Panamá. This HPS episode, resulting in 22% case fatality, was linked to the Costa Rican pigmy rice rat, Oligoryzomys fulvescens costaricensis, which harbored a then undescribed hantavirus, Choclo virus. In addition, Cherrie's cane rat, Zygodontomys brevicauda cherriei, was identified as carrying a distinct hantavirus, Calabazo virus with no known pathogenicity to humans. Herein we present the ecological results of the outbreak investigations in the Azuero region. A total of 164 animals were captured, of which 126 were potential small, non-volant mammal hosts of a hantavirus: rodents in the family Muridae. There were significant differences in small mammal community structure between case sites and a negative control site. Differences were manifest in ecological measures of species diversity and in species evenness and heterogeneity measures, as indicated by Pairwise Euclidian distances and Morisita indices of community similarity. Our analyses suggest that human activities (i.e., deforestation for cattle ranching) coupled with environmental factors (i.e., increased precipitation) may have synergistically coalesced for an increased risk of HPS to area residents.

Hantavirus pulmonary syndrome in the State of São Paulo, Brazil, 1993-1998.

Between 1993 and 1998, 10 cases of clinical hantavirus infection were diagnosed in Brazil. Hantavirus-specific IgM, or positive immunohistochemical analysis for hantavirus antigen, or positive reverse transcription-polymerase chain reaction results for hantavirus RNA were used to confirm nine of these cases; eight were hantavirus pulmonary syndrome (HPS), and one was mild hantavirus disease. The remaining clinical case of hantavirus infection was fatal, and no tissue was available to confirm the diagnosis. During the first 7 months of 1998, five fatal HPS cases caused by a Sin Nombre-like virus were reported from three different regions in the State of São Paulo, Brazil: two in March (Presidente Prudente Region), two in May (Ribeirão Preto Region), and one in July (Itapecerica da Serra Region). Epidemiologic, ecologic, and serologic surveys were conducted among case contacts, area residents, and captured rodents in five locations within the State of São Paulo in June of 1998. Six (4.8%) of 125 case contacts and six (5.2%) of 116 area residents had IgG antibody to Sin Nombre virus (SNV) antigen. No case contacts had a history of HPS-compatible illness, and only one area resident reported a previous acute respiratory illness. A total of 403 rodents were captured during 9 nights of trapping (1969 trap nights). All 27 rodents that were found to be positive for IgG antibody to SNV antigen were captured in crop border and extensively deforested agricultural areas where four of the 1998 HPS case-patients had recently worked. The IgG antibody prevalence data for rodents suggest that Bolomys lasiurus and perhaps Akodon sp. are potential hantavirus reservoirs in this state of Brazil.

Hantavirus pulmonary syndrome in Panama: identification of novel hantaviruses and their likely reservoirs.

Hantavirus pulmonary syndrome (HPS), a severe respiratory disease with high mortality caused by rodent-borne hantaviruses, has previously been identified in the United States and Canada as well as central and southern South America. In late 1999 and early 2000, an outbreak of acute illness compatible with HPS was reported in Los Santos, Panama, with the death of 3 of the 12 (25%) suspected cases. Hantavirus-specific antibodies were detected in patient sera, and virus RNA was detected by reverse transcriptase-polymerase chain reaction. Sequence analysis of virus genome N-, G1-, and G2-encoding fragments showed this to be a novel hantavirus, Choclo virus. Serologic and virus genetic analyses of rodents trapped in the area showed Oligoryzomys fulvescens to be the likely reservoir for the HPS-associated Choclo virus. In addition, Zygodontomys brevicauda rodents were shown to harbor another genetically unique hantavirus, Calabazo virus.

Genetic investigation of novel hantaviruses causing fatal HPS in Brazil.

Although hantavirus pulmonary syndrome (HPS) was discovered in North America in 1993, more recent investigations have shown that the disease is a much larger problem in South America, where a greater number of cases and HPS-associated viruses have now been detected. Here we describe the genetic investigation of three fatal HPS cases from Brazil, including a 1995 case in Castelo dos Sonhos (CAS) in the state of Mato Grosso and two 1996 cases in the counties of Araraquara (ARA) and Franca (FRA), in the state of São Paulo. Reverse transcription-polymerase chain reaction (RT-PCR) products representing fragments of the hantavirus N, G1, and G2 coding regions were amplified from patient acute-phase serum samples, and the nucleotide (nt) sequences (394, 259, and 139 nt, respectively) revealed high deduced amino acid sequence identity between ARA and FRA viruses (99.2%, 96.5%, and 100%, respectively). However, amino acid differences of up to 14.0% were observed when ARA and FRA virus sequences were compared with those of the geographically more distant CAS virus. Analysis of a 643-nt N coding region and a 1734-nt predominantly G2-encoding region of ARA and CAS virus genomes confirmed that these Brazilian viruses were distinct and monophyletic with previously characterized Argentinean hantaviruses, and suggested that Laguna Negra (LN) virus from Paraguay was ancestral to both the Brazilian and Argentinean viruses. The phylogenetic tree based on the N coding fragment also placed LN in a separate clade with Rio Mamore virus from Bolivia. At the amino acid level, ARA and CAS viruses appeared more closely related to the Argentinean viruses than they were to each other. Similarly, analysis of the diagnostic 139-nt G2 fragment showed that the Juquitiba virus detected in a 1993 fatal HPS case close to São Paulo city, Brazil was closer to Argentinean viruses than to ARA or CAS viruses. These data indicate that at least three different hantavirus genetic lineages are associated with Brazilian HPS cases.

Experimental infection of the cane mouse Zygodontomys brevicauda (family Muridae) with guanarito virus (Arenaviridae), the etiologic agent of Venezuelan hemorrhagic fever.

Chronic infections in specific rodents appear to be crucial to the long-term persistence of arenaviruses in nature. The cane mouse, Zygodontomys brevicauda, is a natural host of Guanarito virus (family Arenaviridae), the etiologic agent of Venezuelan hemorrhagic fever. The purpose of this study was to elucidate the natural history of Guanarito virus infection in Z. brevicauda. Thirty-nine laboratory-reared cane mice each were inoculated subcutaneously with 3.0 log10 plaque-forming units of the Guanarito virus prototype strain INH-95551. No lethality was associated with infection in any animal, regardless of age at inoculation. The 13 newborn, 14 weanling, and 8 of the 12 adult animals developed chronic viremic infections characterized by persistent shedding of infectious virus in oropharyngeal secretions and urine. These findings indicate that Guanarito virus infection in Z. brevicauda can be chronic and thus support the concept that this rodent species is the natural reservoir of Guanarito virus.

Clinical case definitions for Argentine hemorrhagic fever.

Argentine hemorrhagic fever (AHF) is a potentially lethal infection in Argentina. The case-fatality ratio is >15%, but treatment reduces the mortality rate to <1%. Diagnosis is based on clinical and laboratory criteria, but no case definition has been validated. A chart review was conducted for patients hospitalized with suspected AHF. Individuals with a fourfold rise in antibody titer were classified as cases. The combination of a platelet count of <100,000/mm3 and a white blood cell (WBC) count of <2,500/mm3 had a sensitivity and specificity of 87% and 88%, respectively, thus suggesting that the use of these criteria in a case definition would be helpful for epidemiological studies of AHF. The combination of a platelet count of <100,000/mm3 and a WBC count of <4,000/mm3 had a sensitivity of 100% and a specificity of 71%; the use of these criteria in a case definition should be helpful for screening patients for therapy with immune plasma in the region where AHF is endemic.

Natural rodent host associations of Guanarito and pirital viruses (Family Arenaviridae) in central Venezuela.

The objective of this study was to elucidate the natural rodent host relationships of Guanarito and Pirital viruses (family Arenaviridae) in the plains of central Venezuela. Ninety-two arenavirus isolates from 607 animals, representing 10 different rodent species, were characterized to the level of serotype. The 92 isolates comprised 19 Guanarito virus strains and 73 Pirital virus strains. The 19 Guanarito virus isolates were from Zygodontomys brevicauda; 72 (98.6%) of the 73 Pirital virus isolates were from Sigmodon alstoni. These results indicate that the natural rodent associations of these 2 sympatric arenaviruses are highly specific and that Z brevicauda and S. alstoni are the principal rodent hosts of Guanarito and Pirital viruses, respectively.

An outbreak of hantavirus pulmonary syndrome, Chile, 1997.

An outbreak of 25 cases of Andes virus-associated hantavirus pulmonary syndrome (HPS) was recognized in southern Chile from July 1997 through January 1998. In addition to the HPS patients, three persons with mild hantaviral disease and one person with asymptomatic acute infection were identified. Epidemiologic studies suggested person-to-person transmission in two of three family clusters. Ecologic studies showed very high densities of several species of sigmodontine rodents in the area.

Assessment of person-to-person transmission of hantavirus pulmonary syndrome in a Chilean hospital setting.

Person-to-person transmission of Andes hantavirus among healthcare workers was reported in Argentina for the first time in 1996. To determine whether such transmission of the virus occurred during a 1997 outbreak of hantavirus pulmonary syndrome (HPS) in southern Chile due to Andes virus, we conducted a serological and epidemiological study in the Coyhaique Regional Hospital, where the majority of HPS patients were admitted. Workers in every department of the hospital were evaluated for immunoglobulin G (IgG) and IgM hantavirus antibodies using the enzyme-linked immunosorbent assay (ELISA). A standardized questionnaire was used to determine the type and extent of exposure to HPS patients, as well as other potential risk factors and previous febrile respiratory illnesses requiring hospitalization. Less than half (44%) reported always using gloves when touching patients or their secretions; respiratory protection was not used. Of the 319 participants (87.9% of those eligible), 12 (3.7%) had IgG antibodies. This finding is consistent with the antibody prevalence in the community in which the participants lived. Of the 12 positive healthcare workers, six reported contact with HPS patients. A similar exposure was found in those who tested negative [6/140 (4%) compared to 6/179 (3%), P = 0.66]. There was no significant difference in the types of hospital activities performed or the number of hospitalizations for febrile respiratory illnesses between antibody-positive and antibody-negative individuals. These data suggest that there was no person-to-person transmission among healthcare workers during a recent outbreak of HPS in Southern Chile in 1997, despite the inconsistent use of any precautions against transmission.

Venezuelan hemorrhagic fever: clinical and epidemiological studies of 165 cases.

Epidemiological and clinical data are presented on 165 cases of Venezuelan hemorrhagic fever (VHF), a newly emerging viral zoonosis caused by Guanarito virus (of the family Arenaviridae). The disease is endemic in a relatively circumscribed area of central Venezuela. Since its first recognition in 1989, the incidence of VHF has peaked each year between November and January, during the period of major agricultural activity in the region of endemicity. The majority of cases have involved male agricultural workers. Principal symptoms among the patients with VHF included fever, malaise, headache, arthralgia, sore throat, vomiting, abdominal pain, diarrhea, convulsions, and a variety of hemorrhagic manifestations. The majority of patients also had leukopenia and thrombocytopenia. The overall fatality rate among the 165 cases was 33.3%, despite hospitalization and vigorous supportive care.

Protective efficacy of a live attenuated vaccine against Argentine hemorrhagic fever. AHF Study Group.

Argentine hemorrhagic fever (AHF), caused by the arenavirus Junin, is a major public health problem among agricultural workers in Argentina. A prospective, randomized, double-blind, placebo-controlled, efficacy trial of Candid 1, a live attenuated Junin virus vaccine, was conducted over two consecutive epidemic seasons among 6500 male agricultural workers in the AHF-endemic region. Twenty-three men developed laboratory-confirmed AHF during the study; 22 received placebo and 1 received vaccine (vaccine efficacy 95%; 95% confidence interval [CI], 82%-99%). Three additional subjects in each group developed laboratory-confirmed Junin virus infection associated with mild illnesses that did not fulfill the clinical case definition for AHF, yielding a protective efficacy for prevention of any illness associated with Junin virus infection of 84% (95% CI, 60%-94%). No serious adverse events were attributed to vaccination. Candid 1, the first vaccine for the prevention of illness caused by an arenavirus, is safe and highly efficacious.

Laguna Negra virus associated with HPS in western Paraguay and Bolivia.

A large outbreak of hantavirus pulmonary syndrome (HPS) recently occurred in the Chaco region of Paraguay. Using PCR approaches, partial virus genome sequences were obtained from 5 human sera, and spleens from 5 Calomys laucha rodents from the outbreak area. Genetic analysis revealed a newly discovered hantavirus, Laguna Negra (LN) virus, to be associated with the HPS outbreak and established a direct genetic link between the virus detected in the HPS cases and in the C. laucha rodents, implicating them as the primary rodent reservoir for LN virus in Paraguay. Virus isolates were obtained from two C. laucha, and represent the first successful isolation of a pathogenic South American hantavirus. Analysis of the prototype LN virus entire S and M and partial L segment nucleotide and deduced amino acid sequences showed that this virus is unique among the Sigmodontinae-borne clade of hantaviruses. Analysis of PCR fragments amplified from a serum sample from a Chilean HPS patient, who had recently traveled extensively in Bolivia (where C. laucha are known to occur), revealed an LN virus variant that was approximately 15% different at the nucleotide level and identical at the deduced amino acid level relative to the Paraguayan LN virus. These data suggest that LN virus may cause HPS in several countries in this geographic region.

Secreted proteophosphoglycan of Leishmania mexicana amastigotes activates complement by triggering the mannan binding lectin pathway.

Cutaneous lesions induced by infection of mice with the protozoan parasite, Leishmania mexicana, contain abundant amounts of a high molecular mass proteophosphoglycan (PPG), which is secreted by the amastigote stage residing in phagolysosomes of macrophages and can then be released into the tissue upon rupture of the infected cells. Amastigote PPG forms sausage-shaped but soluble particles and belongs to a novel class of serine-rich proteins that are extensively O-glycosylated by phosphooligosaccharides capped by mannooligosaccharides. The purified molecule is shown here to efficiently activate complement (C) and deplete hemolytic activity of normal serum and may prevent the opsonization of L. mexicana amastigotes. Complement activation is Ca2+ dependent but does not depend on antibodies or the complement component C1. PPG binds to serum mannan binding protein (MBP), thus activating the MBP-associated serine protease, P100. Subsequently, the C cascade is triggered through C4 leading to covalent modification probably of carbohydrate hydroxyls of PPG by C3 fragments. Thus, PPG is able to activate C via the mannan binding lectin pathway which is unusual for secreted, soluble products of microbial origin. The proteophosphoglycan-induced complement activation is postulated to contribute to the lesion development and pathology caused by the parasite.

An outbreak of hantavirus pulmonary syndrome in western Paraguay.

During an investigation of hantavirus pulmonary syndrome (HPS) in Paraguay in 1995, sera from persons with HPS-like illness, houshold contacts of confirmed HPS case-patients, and a sample of the area residents were analyzed by ELISA for antibodies to Sin Nombre virus (SNV). Rodent serosurveys and analysis of precipitation records were also conducted. Twenty-three of 24 available probable cases were SNV antibody-positive, 17 of whom were ill between July 1995 and January 1996. Four (14.8%) of 27 case-contacts and 44 (12.8%) of 345 community residents were also seropositive. Calomys laucha (vesper mouse) was the most common rodent species captured and the most frequently SNV-seropositive. Rainfall in May 1995 was 10-fold greater than that seen in May over the preceding 11 years. This 17 case-cluster represents the largest documented outbreak since HPS was first recognized in 1993. Calomys laucha is the likely primary rodent reservoir for a SNV-like hantavirus in western Paraguay. Fluctuations in monthly precipitation rates may have contributed to increased risk for HPS in this region.