Technical Appendix Research Articles

Novel Hepatitis E Virus in Ferrets, the Netherlands

To the Editor: Hepatitis E virus (HEV), a member of the family Hepeviridae and the genus Hepevirus, is transmitted by the fecal–oral route and causes liver inflammation, which leads to mortality rates of ≤20% in pregnant woman (1,2). Human hepatitis E is a major disease not only in developing countries but also in industrialized countries, and identification of animal strains of HEV in pigs and deer and its zoonotic potential has raised considerable public health concerns (1,3). Recent reports suggest that other animals such as rats, mongooses, chickens, rabbits, and trout also may harbor HEVs (1–5). The genomes of these viruses are ≈6.6 kb–7.2 kb and encode 3 open reading frames (ORFs) flanked by a capped 5′ end and a poly A tail at the 3′ end (1,3). We used random PCR amplification and high-throughput sequencing technology to investigate HEV sequences in ferrets (Mustela putorius) from the Netherlands. In 2010, fecal samples were collected from ferrets in the Netherlands and stored at −80°C. Samples that were negative for ferret coronavirus (6) were further characterized for other pathogens. Viral RNA was isolated and viral metagenomic libraries were constructed for 454 pyrosequencing as described (7,8), and 248,840 sequence reads were generated from 7 fecal samples. Using Blastn and Blastx (www.ncbi.nlm.nih.gov/BLAST), we identified 289 sequence reads in 1 sample that were related to rat HEV and that could be assembled into 6 contigs covering ≈50% of the ferret HEV (FRHEV) genome. We then developed a set of nested PCR primers on the basis of obtained sequences to detect viral RNA (Technical Appendix Table 1). Total RNA extracted from 43 ferret fecal samples collected from 19 locations in the Netherlands was used to perform reverse transcription PCR amplification. Using this PCR, we detected viral RNA in 4 (9.3%) fecal samples tested from 4 locations (distance between each sampling location ranged from 25 km to 127 km). All amplicons were confirmed by nucleotide sequencing. We have limited information regarding the clinical disease this virus may cause because these samples were obtained from household pet ferrets that did not show overt clinical signs. In addition, 4/16 animals from a single farm were IgG positive when tested for IgG against HEV by using recombinant human HEV protein (Wantai, Beijing, China). To characterize the complete genome of this virus, we selected 2 PCR-positive samples (FRHEV4 and FRHEV20), developed different sets of specific primers on the basis of sequence fragments obtained by 454 pyrosequencing, directly sequenced amplicons by Sanger sequencing, and used a rapid amplification of cDNA ends PCR to obtain 5′ and 3′ frame end sequences. Using overlapping fragments we assembled 2 complete FRHEV genome sequences that contained 6,854 nt, including a 13-nt 3′ poly A tail and a 12-nt 5′ end. FRHEV full-genome sequences FRHEV4 and FRHEV20 showed 98.6% sequence identity and were deposited into GenBank under accession nos. {type:entrez-nucleotide,attrs:{text:JN998606,term_id:397310723,term_text:JN998606}}JN998606 and {type:entrez-nucleotide,attrs:{text:JN998607,term_id:397310728,term_text:JN998607}}JN998607, respectively. The FRHEV genome contains a complete ORF1 gene that encodes a nonstructural protein of 1,596 aa, an ORF2 gene that encodes a capsid protein of 654 aa, an ORF3 gene that encodes a phosphoprotein of 108 aa, and a 3′ noncoding region of 78 nt. Sequence analyses indicated that the FRHEV genome shared the highest identity (72.3%) with rat HEV. Sequence identity with HEV genotypes 1–4 and rabbit and avian HEVs ranged from 54.5% to 60.5% (Technical Appendix Table 2). The FRHEV genome organization was found to be slightly different from other HEVs and included a putative ORF (ORF4) of 552 nt that overlapped with ORF1 (Technical Appendix Figure). A similar pattern of genome organization was observed for both FRHEVs. Phylogenetic analysis of the complete genomes clearly showed that FRHEV was separated from genotype 1–4 HEVs and clustered with rat HEV (Figure). Similar phylogenetic clustering was observed when nucleotide and deduced amino acid sequences of ORF1, ORF2, and ORF3 were analyzed separately. The phylogenetic distance between rat HEV and FRHEV is larger than the distance between genotype 1 and genotype 2 HEV. Figure Phylogenetic tree based on the complete genomic sequences of ferret hepatitis E viruses (HEVs) and human, rabbit, swine, avian, and rat HEV strains. Names of HEV strains follow GenBank accession numbers. Sequence alignment was performed by using ClustalW ... In recent years, an increasing number of sporadic cases of hepatitis E have been reported (1,9). Several observations suggest that autochthonous cases are caused by zoonotic spread of infection from wild or domestic animals (1,3,9). In addition, IgG anti-HEV seropositivity in the United States has been associated with several factors, including having a pet at home (10). Further studies are needed to identify the zoonotic potential of FRHEV. Technical Appendix: Genome organization of hepatitis E viruses (HEVs) and initiation of translation of open reading frame 1 (ORF1), ORF2, and ORF3 of ferret HEV. Click here to view.(190K, pdf)

Usefulness of School Absenteeism Data for Predicting Influenza Outbreaks, United States

Usefulness of School Absenteeism Data for Predicting Influenza Outbreaks, United States

Carpal Tunnel Syndrome with Paracoccidioidomycosis

Carpal Tunnel Syndrome with Paracoccidioidomycosis

Factors Influencing Emergence of Tularemia, Hungary, 1984–2010

To the Editor: Francisella tularensis, the etiologic agent of tularemia, is a highly infectious zoonotic agent. F. tularensis subsp. holarctica (type B) is found throughout the Northern Hemisphere and is the only subspecies found in Europe (1). Lagomorphs and rodents probably serve as the primary mammalian reservoir hosts, and hematophagous arthropods, such as ticks, play a role as vectors and hosts (2,3). Although F. tularensis is a potential agent of biological warfare and several emergences and reemergences of tularemia have been reported around the world (1,4), the epizootiology of the disease is only partially understood. The aim of our study was to analyze factors that influence the emergence of tularemia in Hungary. The study area (15,475 km2) included 3 counties in eastern Hungary. The analyzed data represented a period of 25 years, March 1984–February 2010. Annual F. tularensis–specific seroprevalence data for the European brown hare (Lepus europaeus) population were obtained by slide agglutination testing during the winter (December and January) screening of 2,500–25,000 animals (Technical Appendix). Population density data (animals/km2) for hares were based on February line transect counts and were obtained from the Hungarian Game Management database (www.vvt.gau.hu/vadgazdalkodasi_statisztikak.htm). Common vole (Microtus arvalis) densities (calculated from the number of active burrows/hectare during November) for 1996–2010 were obtained from the Central Agriculture Office, Budapest, Hungary. Vole density was scaled from 0 (absent) to 10 (peak population). The annual number of tularemia cases in humans (based on clinical history and tube agglutination test results) was obtained from the National Center for Epidemiology, Budapest. The data were regrouped according to the yearly biologic cycle (March–February) for hares and voles (Figure), and relationships between these yearly data were quantified by using the Spearman rank correlation coefficient (5) at county and regional levels. A 2–3 year cycle was characteristic for the analyzed data. A significant positive correlation was found among the number of tularemia cases in humans and the seroprevalence of F. tularensis among European brown hares (Spearman ρ = 0.73; p<0.0001) and the population density of common voles (Spearman ρ = 0.77; p = 0.0081). A significant negative correlation was found between the population density of hares and the seroprevalence of F. tularensis in hares (Spearman ρ = −0.41; p = 0.0365). Figure Correlation between the seroprevalence of Francisella tularensis in the European brown hare (Lepus europaeus) population, the population density of European brown hares and common voles (Microtus arvalis), and the number of tularemia cases in humans eastern ... The comprehensive and long-term annual data used in this study provide clues regarding the factors shaping the intraannual epizootiology and emergence or reemergence of tularemia. The European brown hare is moderately sensitive to F. tularensis subsp. holarctica. The hares produce a heterogeneous response to infection, which means that some die of overwhelming bacteremia and others survive with a protracted course of infection, thereby contributing to the maintenance of tularemia over longer periods and serving as useful sentinels of disease activity. Other studies have concluded that hares, together with infected ticks, may serve as disease reservoirs between epizootics (2,3,6,7). However, we instead hypothesize that the 2–3 year cycling feature of the population dynamics for the common vole (2) determines the ecology of F. tularensis subsp. holarctica in eastern Hungary. The common vole is highly susceptible to F. tularensis subsp. holarctica (3,8). When population densities among voles are high, F. tularensis disease transmission and spillover to hares may be facilitated by stress-related aggression, cannibalism, and F. tularensis contamination of the environment by infectious body discharges (2). Enhanced transmission and spillover can expand local outbreaks to epizootic proportions, and infected hares may, in turn, further enhance the spread of disease through bacterial shedding in urine (6,7). The disease in hares often results in septicemia and death (7), thus decreasing the population density of these animals. Hares and especially voles are also hosts for different stages of several tick species (2,6), so it can be expected, that higher numbers of infected rodents and lagomorphs result in an increased proportion of infected ticks and, thus, increased transmission of F. tularensis subsp. holarctica. It can be concluded that a higher number of infection sources in the environment results in elevated numbers of cases in humans, mainly through the handling and skinning of hares, but also through tick bites and, potentially, the inhalation of infectious aerosols originating from, for example, hay or grain. Technical Appendix: Detailed data of the annual Francisella tularensis–specific seroprevalence in the European brown hare (Lepus europaeus) population, the annual population density of European brown hares and common voles (Microtus arvalis), and the annual number of tularemia cases in humans, Hungary, 1984–2010. Data are for 3 counties in eastern Hungary: Bekes, Csongrad, and Jasz-Nagykun-Szolnok)*. Click here to view.(47K, xls)

2011 Review of Conditionality - Technical Appendices

This section provides the background studies relating to dimensions of Fund policy on conditionality. Appendix 1 provides a review of Fund experience with coordination, both in a low-income country (LIC) setting (in African programs) and in an emerging market and advanced economy setting in the European Union (EU) and Euro Area (EA). Appendix 2 summarizes the recent changes to debt limits in LICs and provides an assessment of the implementation of this policy in the early stages (up to mid-February 2011). Appendix 3 reviews the experience of countries with the Flexible Credit Line (FCL) and Precautionary Credit Line (PCL)-supported programs. Appendix 4 examines the impact of the 2009 Special Drawing Rights (SDR) allocation on program design

Zoonotic Disease Pathogens in Fish Used for Pedicure

Zoonotic Disease Pathogens in Fish Used for Pedicure

NovelChlamydiaceaeDisease in Captive Salamanders

To the Editor: Although 2 major diseases of amphibians, chytridiomycosis and ranavirosis, have been relatively well studied, enigmatic amphibian disease and death not attributable to any of the known amphibian diseases frequently occur (1). We describe an apparently new disease in salamanders that is associated with a novel genus within the family Chlamydiaceae. The salamanders seen in our clinic belonged to 1 of the following species: Salamandra corsica, the Corsican fire salamander (5 animals from 1 collection); Neurergus crocatus, the yellow spotted newt (11 animals from 3 collections); or N. strauchii, Strauch’s spotted newt (6 animals from 2 collections). All salamanders were captive bred; housed in breeding colonies in private collections in Elsloo and Eindhoven, the Netherlands, Munich, Germany, and Brugge, Belgium; and 1–3 years of age. Disease was characterized by anorexia, lethargy, edema, and markedly abnormal gait. Mortality rate was 100%. Animals in these collections had no histories of disease. All animals were in good nutritional condition. Necropsy did not yield any macroscopic lesions. All animals had mild intestinal nematode or protozoan infections. Results of real-time PCRs for iridoviruses in liver and skin (2) or Batrachochytrium dendrobatidis fungus of skin (3) were negative for all animals. We placed liver suspensions from the dead salamanders on Columbia agar with 5% sheep blood and tryptic soy agar and then incubated the samples up to 14 days at 20°C. No consistent bacterial growth was observed. Histologic examination of 2 Corsican fire salamanders and 1 yellow spotted newt revealed hepatitis in 1 of the Corsican fire salamanders and the yellow spotted newt. Hepatitis was characterized by high numbers of melanomacrophages and a marked infiltration of granulocytic leukocytes. Immunohistochemical staining for chlamydia (IMAGEN Chlamydia; Oxoid, Basingstone, UK) showed cell-associated fluorescently stained aggregates in liver tissue, suggestive of Chlamydiales bacteria. Transmission electron microscopic examination of the liver of a yellow spotted newt revealed intracellular inclusions containing particles matching the morphology of reticulate or elementary bodies of Chlamydiaceae (Technical Appendix). A PCR (4) to detect the 16S rRNA of all Chlamydiales bacteria, performed on liver tissue samples from all animals, yielded positive results in all 5 Corsican fire salamanders; in 4/7, 1/3, and 1/1 yellow spotted newts; and in 4/5 and 1/1 Strauch’s spotted newts. For taxon identification, the 16S rRNA gene of the Chlamydiales bacteria was amplified and sequenced from the livers from 2 yellow spotted newts (1 from the collection in Elsloo, the Netherlands and 1 from the collection in Munich, Germany), 1 Strauch’s spotted newt, and 5 Corsican fire salamanders. The sequences shared >90% nt identity with the 16S rRNA gene of C. abortus B577 (GenBank accession no. {type:entrez-nucleotide,attrs:{text:D85709,term_id:2114161,term_text:D85709}}D85709) and therefore can be identified as a member of the family Chlamydiaceae (5). The closest 16S rRNA similarity (92%) was observed with C. psittaci strain CPX0308 ({type:entrez-nucleotide,attrs:{text:AB285329,term_id:148537245,term_text:AB285329}}AB285329). The sequence obtained from all spotted newt species specimens was identical (GenBank accession no. {type:entrez-nucleotide,attrs:{text:JN392920,term_id:358009359,term_text:JN392920}}JN392920) but differed slightly (1%) from that obtained from the fire salamander species specimens (GenBank accession no. {type:entrez-nucleotide,attrs:{text:JN392919,term_id:358009358,term_text:JN392919}}JN392919). These sequence differences point to the existence of multiple strains with possible host adaptation. We determined the phylogenetic position of the novel taxon, named Candidatus Amphibiichlamydia salamandrae (Technical Appendix), identified by using neighbor-joining analysis with Kodon software (Applied Maths, Sint-Martens-Latem, Belgium). The novel Chlamydiales forms a distinct branch in the well-supported monophyletic clade with the genera Chlamydia and Candidatus Clavochlamydia salmonicola (family Chlamydiaceae) (Figure). Maximum parsimony and unweighted pair group with arithmetic mean analyses yielded cladograms with the same topology (results not shown). Previous reports of members of the family Chlamydiaceae in amphibians concerned species occurring in other vertebrate taxa as well: C. psittaci, C. pneumoniae, C. abortus, and C. suis (6–10). To our knowledge, this member of the family Chlamydiaceae has been seen in amphibians, but not in other vertebrate hosts. The 16S rRNA analysis showed this taxon to belong to a clade with Candidatus Clavochlamydia salmonicola, a taxon found in fish. The phylogenetic position of the novel taxon in the family Chlamydiaceae thus roughly reflects the phylogenetic relation between the host species, providing evidence for host–bacterium co-evolution in the family Chlamydiaceae. Figure Topology of the novel amphibian Chlamydiaceae (Candidatus Amphibiichlamydia salamandrae) within the phylogenetic tree obtained by neighbor-joining and based on 16S rRNA gene data from representative species. Numbers show the percentage of times each branch ... Although the results obtained are not conclusive with regard to the pathogenic potential of this novel genus and species of Chlamydiales, we were not able to attribute the clinical signs to any known disease. We therefore suggest that we discovered a novel bacterial taxon with possible considerable impact on amphibian health.

Getting it wrong: how faulty monetary statistics undermine the Fed, the financial system, and the economy

Blame for the recent financial crisis and subsequent recession has commonly been assigned to everyone from Wall Street firms to individual homeowners. It has been widely argued that the crisis and recession were caused by “greed” and the failure of mainstream economics. In Getting It Wrong, leading economist William Barnett argues instead that there was too little use of the relevant economics, especially from the literature on economic measurement. Barnett contends that as financial instruments became more complex, the simple-sum monetary aggregation formulas used by central banks, including the U.S. Federal Reserve, became obsolete. Instead, a major increase in public availability of best-practice data was needed. Households, firms, and governments, lacking the requisite information, incorrectly assessed systemic risk and significantly increased their leverage and risk-taking activities. Better financial data, Barnett argues, could have signaled the misperceptions and prevented the erroneous systemic-risk assessments. When extensive, best-practice information is not available from the central bank, increased regulation can constrain the adverse consequences of ill-informed decisions. Instead, there was deregulation. The result, Barnett argues, was a worst-case toxic mix: increasing complexity of financial instruments, inadequate and poor-quality data, and declining regulation. Following his accessible narrative of the deep causes of the crisis and the long history of private and public errors, Barnett provides technical appendixes, containing the mathematical analysis supporting his arguments.

Recognition and Diagnosis ofCryptococcus gattiiInfections in the United States

Recognition and Diagnosis of<i>Cryptococcus gattii</i>Infections in the United States

Does Indivisible Labor Explain the Difference between Micro and Macro Elasticities? A Meta-Analysis of Extensive Margin Elasticities

Macroeconomic calibrations imply much larger labor supply elasticities than microeconometric studies. One prominent explanation for this divergence is that indivisible labor generates extensive margin responses that are not captured in micro studies of hours choices. We evaluate whether existing calibrations of macro models are consistent with micro evidence on extensive margin responses using two approaches. First, we use a standard calibrated macro model to simulate the impacts of tax policy changes on labor supply. Second, we present a metaanalysis of quasi-experimental estimates of extensive margin elasticities. We find that micro estimates are consistent with macro evidence on the steady-state (Hicksian) elasticities relevant for cross-country comparisons. However, micro estimates of extensive-margin elasticities are an order of magnitude smaller than the values needed to explain business cycle fluctuations in aggregate hours. Hence, indivisible labor supply does not explain the large gap between micro and macro estimates of intertemporal substitution (Frisch) elasticities. Our synthesis of the micro evidence points to Hicksian elasticities of 0.3 on the intensive and 0.25 on the extensive margin and Frisch elasticities of 0.5 on the intensive and 0.25 on the extensive margin.

Enhancing impact: visualization of an integrated impact assessment strategy

The environmental impact assessment process is over 40 years old and has dramatically expanded. Topics, such as social, health and human rights impact are now included. The main body of an impact analysis is generally hundreds of pages long and supported by countless technical appendices. For large, oil/gas, mining and water resources projects both the volume and technical sophistication of the reports has far exceeded the processing ability of host communities. Instead of informing and empowering, the reports are abstruse and overwhelming. Reinvention is required. The development of a visual integrated impact assessment strategy that utilizes remote sensing and spatial analyses is described.

Claire L Wendland. A Heart for the Work: Journeys through an African Medical School. Chicago: University of Chicago Press, 2010. xiv + 330 pp. Technical Appendix. Notes. Glossary. Bibliography. Index. $27.50. Paper.

HEALTH AND DISEASE Claire L. Wendland. A Heart for the Work: Journeys through an African Medical School. Chicago: University of Chicago Press, 2010. xiv + 330 pp. Technical Appendix. Notes. Glossary. Bibliography. Index. $27.50. Paper. Claire Wendland provides a compelling account of medical training in Africa in A Heart for the Work. In her analyses of the socialization of MaIawian physicians and the practice of African biomedicine, Wendland makes a valuable contribution to the literature on medical power/knowledge, particularly in poor and postcolonial contexts, and provides an all too rare example of anthropology studying up in Africa. Herself a Northerntrained obstetrician-gynecologist and anthropologist, Wendland uses her case to argue that biomedical professionals everywhere are shaped as much by the social, cultural, and material conditions in which they train and practice as by the characteristics of biomedicine in hegemonic form. This coproduction, then, has important consequences for individual physicians and, through their medical practice, for their patients and their nation. Wendland writes accessibly to distill complicated social theory, and biomedical knowledge and experience, into an effordess prose that most any reader could easily engage with. The book deserves to be read far and wide: while Wendland clearly writes with her undergraduate anthropology, sociology, and premedicai students in mind, professionals in medical education, international public health and development, and international public administration broadly will also find a trove of useful analysis and insight. Scholars of biomedicine and scientific expertise will find the case of Malawian physicians a compelling counterpoint to the Eurocentric literature. Finally, as Wendland keeps the contemporary history and context of southern Africa at the forefront of her work, her analysis of modern African biomedicine is useful for Africanists of all stripes. The book is cogendy arranged in seven chapters that follow the progress of medical students through their course of study at the University of Malawi's College of Medicine. Wendland's data comes from a year of intensive fieldwork (2002-3) combined with shorter trips before and after; her view of student progress through the training program is thus crosssectional rather than longitudinal. Instead of following one cohort through the five-year program and beyond, Wendland creates comparative snapshots of different cohorts at various stages of becoming competent physicians. This organization allows her to correlate students' experience with historical phenomena as well as with changing policies in the university system. …

On the theory of core-mantle coupling

This article commences by surveying the basic dynamics of Earth's core and their impact on various mechanisms of core-mantle coupling. The physics governing core convection and magnetic field production in the Earth is briefly reviewed. Convection is taken to be a small perturbation from a hydrostatic, “adiabatic reference state” of uniform composition and specific entropy, in which thermodynamic variables depend only on the gravitational potential. The four principal processes coupling the rotation of the mantle to the rotations of the inner and outer cores are analyzed: viscosity, topography, gravity and magnetic field. The gravitational potential of density anomalies in the mantle and inner core creates density differences in the fluid core that greatly exceed those associated with convection. The implications of the resulting “adiabatic torques” on topographic and gravitational coupling are considered. A new approach to the gravitational interaction between the inner core and the mantle, and the associated gravitational oscillations, is presented. Magnetic coupling through torsional waves is studied. A fresh analysis of torsional waves identifies new terms previously overlooked. The magnetic boundary layer on the core-mantle boundary is studied and shown to attenuate the waves significantly. It also hosts relatively high speed flows that influence the angular momentum budget. The magnetic coupling of the solid core to fluid in the tangent cylinder is investigated. Four technical appendices derive, and present solutions of, the torsional wave equation, analyze the associated magnetic boundary layers at the top and bottom of the fluid core, and consider gravitational and magnetic coupling from a more general standpoint. A fifth presents a simple model of the adiabatic reference state.

Shared Human/Rabbit Ligands for Rabbit Hemorrhagic Disease Virus

To the Editor: Rabbit hemorrhagic disease virus (RHDV) is a calicivirus of the genus Lagovirus that causes epidemics of an acute disease and mortality rates of 50%–90% among rabbits. The disease, which was first described in 1984, is characterized by hemorrhagic lesions, mainly affecting the liver and lungs 24–72 h after infection (1). Similar to human caliciviruses of the genus Norovirus, RHDV binds to histo-blood group antigens (HBGAs), and we recently showed that HBGAs serve as attachment factors (ligands) that facilitate RHDV infection (2). HBGAs are polymorphic carbohydrate structures representing terminally exposed portions of larger glycans linked to proteins or glycolipids. In many vertebrate species, they are mainly expressed on epithelial surfaces. Because phylogenetic conservation of receptors is a major risk factor for cross-species transmission (3), we analyzed the ability of RHDV strains to recognize human HBGAs expressed on epithelia. We obtained 38 saliva samples from healthy persons with ABO, Secretor, and Lewis phenotypes, and we selected confirmed FUT2 (secretor) and FUT3 (Lewis) genotypes (4) to include ABO, secretor, and Lewis phenotypic diversity. Binding capacity of 6 RHDV strains representative of virus diversity (2) was tested against human saliva samples by using a method similar to that reported for human norovirus (5). In brief, saliva samples diluted 1:1,000 or B type 2 bovine serum albumin–conjugated tetrasaccharide (positive control) were coated on ELISA plates. After blocking with milk diluted in phosphate-buffered saline, RHDV strains isolated from whole liver extracts of infected animals were incubated on coated plates at dilutions corresponding to 1 × 109 genome copies (0.2 µg/mL capsid protein equivalent) as determined by Nystrom et al. (2). Monoclonal antibody 2G3, biotinylated anti-mouse IgG, and peroxidase-conjugated avidin were used for RHDV detection; 3,3′,5,5′-tetramethylbenzidine was used as a substrate; and optical density values at 450 nm were measured (2). Binding to the B type 2 epitope was observed for all 6 strains (Technical Appendix Figure, panel A). Human saliva samples were recognized by 5 of 6 RHDV strains. Only G6, an RHDV antigenic variant also known as RHDVa (6), did not show binding to saliva. Strains G1 and G2 showed preferential binding to saliva from B secretors over that from O secretors, and A secretors were poorly recognized. Better recognition of A secretor saliva was obtained with the G3 strain. The G4 and G5 strains showed a clear preference for A secretors over B and O secretors, which indicated a shift in specificity toward recognition of the A antigen from the H and B antigens, as reported (2). None of the strains recognized nonsecretor saliva, which showed that binding to human saliva required A, B, or H motifs. This finding was confirmed by drastically decreased binding after removal of A, B, and H epitopes from secretor saliva by treatment with specific glycosidases. There was no relationship with the Lewis status. To determine if human epithelial cells were recognized by RHDV, binding of the G3 strain to human tissue sections was assessed. Human trachea, lung, and gastroduodenal junction samples obtained from organ donors (before current French restrictions of December 1988) were used to prepare tissue microarrays. Tissues from 18 persons were used and represented the following phenotypes: O secretor Lewis+ (n = 8), A secretor Lewis+ (n = 3), B secretor Lewis+ (n = 2), O secretor Lewis– (n = 1), and O nonsecretor Lewis+ (n = 4). Deparafinated and endogen peroxidase–blocked sections were incubated overnight at 4°C with the G3 strain from an infected liver extract at a concentration of 2 × 109 genome copies/mL. Binding was detected by using monoclonal antibody 2G3 against RHDV, biotinylated anti-mouse IgG, horseradish peroxidase–conjugated avidin, and 3-amino-9-ethylcarbazole substrate with hemalum counterstaining, as described (2). Staining of epithelial cells of stomach or trachea of secretors, but not those of nonsecretors, was observed. This finding indicated that attachment factors for RHDV are present on human cells that constitute potential points of entry for RHDV (Technical Appendix Figure, panels B–E). Attachment to HBGAs of human calicivirus strains represents a first step of the infection process (7). In this study, we have shown that cross-species recognition of HBGAs in cells that may be likely points of entry of RHDV into human cells. RHDV infection has been shown to be rabbit specific (8), which indicates that other molecular elements not shared by rabbits and other mammals restrict its host range. Nevertheless, RHDV RNA was recently isolated from sympatric wild small mammals, which suggested that the species range of RHDV may not be as limited as previously believed (9). In addition, recent phylogenetic analysis showed that caliciviruses exhibit high levels of host switching (10). Therefore, surveillance of RHDV and studies to decipher molecular mechanisms involved in its extreme pathogenicity are warranted.

Serologic Evidence of Nipah Virus Infection in Bats, Vietnam

To the Editor: Bats are potential reservoir for highly pathogenic viruses, such as Nipah virus (NiV) and Hendra virus, which can cross species barriers (1). However, only limited surveillance has been conducted to assess risk for infection by these deadly emerging viruses. We conducted a study in Vietnam from 2007 to 2008 to assess the prevalence of these pathogens in bats. Different species of live bats were obtained from hunters or were captured in caves, pepper fields, and residential areas by using mist nets or harp traps (Technical Appendix Figure 1). A total of 451 serum samples were collected and subjected to IgG ELISA by using an Escherichia coli–derived recombinant nucleocapsid (N) protein of NiV (NiV-N-ELISA) (2). Two Leschenault’s rousette bats (Rousettus leschenaulti) were vaccinated with the same recombinant N protein to obtain positive serum specimens that contained antibodies against NiV-N. An optical density of 492 nm for negative control serum (1,000× dilution) was designated as 1:1,000 ELISA units. ELISA titers of sample serum specimens were obtained at a single dilution (1,000×) by using a standard curve of positive serum with high titers. A sample titer >3,000 was considered positive for IgG against NiV. Positive results were detected from only 2 fruit bat species, R. leschenaulti (31 bats [49.1%]) and Cynopterus sphinx (3 bats [2.8%]) (Table). Of the 34 samples positive by ELISA, only 22 (20 from the former and 2 from the latter bat species), which had enough volume left, were further analyzed by Western blot (WB) with E. coli–expressed recombinant N protein of NiV (Table; Technical Appendix Figure 2). Twenty of the 22 specimens were confirmed as positive by WB. ELISA-positive samples with high titers were also positive by WB for both bat species. However, only 1 sample from an R. leschenaulti bat was positive by WB that used a baculovirus-expressed recombinant N protein. Because of the different protein expression systems, the reactivity of bat antibody against NiV protein in WBs showed different patterns (Technical Appendix Figure 2). Neutralization tests (NTs) in which live NiVs (strain Ma-JMR-01–98) were used were performed at the Institute of Tropical Medicine, Nagasaki University. No specimens of C. sphinx bats were positive by NT; however, 2 specimens from R. leschenaulti bats, both positive with low titers, were confirmed to be positive by NT (50% cytopathic effect after NT, titers of 33.6 and 14.1). However, the latter specimen was negative by WB analysis. Table Results of serologic tests for Nipah virus on bats captured in Vietnam, 2007–2008* Seroepidemiologic studies in other countries have indicated that Pteropus spp. bats (fruit-eating bats) are the main reservoirs for NiV (3–6). Pteropid bats are usually found only in southern Vietnam. We could not obtain these bats for our study. However, a relatively high prevalence (49.1%) of henipavirus antibody was found in R. leschenaulti specimens from Hoa Binh Province. Rousettus spp. bats are the only megabats that use echolocation. These bats hang together on cave ceilings in a tightly packed manner and in groups composed of bats of both sexes and of different ages. They roost in large colonies and fly vast distances to find fruit (7). This behavior might be related to their high rate of seropositivity for viral infections. In southern China, bats of the same species showed a high prevalence of henipavirus antibody (8). R. leschenaulti bats are distributed from central to northern Vietnam and southern China. C. sphinx bats are common all over Vietnam, and their habitat overlaps with that of pteropid bats in southern Vietnam. Previous studies showed that IgG ELISA results for NiV-positive flying foxes correlated well with NT results (3,4). However, in our study, discrepancies existed between NT results and NiV-N-ELISA and WB results. A reason for these differences could be that Nipah-like viruses are circulating among bats in Vietnam, producing antibodies that are cross-reactive by ELISA and WB, but poorly cross-reactive by NT. The cross-reactive antibodies were probably not directed against neutralizing epitopes. To date, no reports have been made of an increased number of febrile encephalitis cases among the residents in Hoa Binh and Dak Lak Provinces where seropositive bats were captured. The circulating viruses may lack the pathogenic potential of Hendra and Nipah viruses. A survey by questionnaire was conducted among residents of Dak Nong and Dak Lak Provinces, where NiV-N-ELISA–positive C. sphinx bats were captured, to determine the frequency of contact between humans and bats. Risk factors for infection were observed in this study, such as bat hunting and cooking and drinking bat blood. In such situations, persons have direct contact with bat body fluids and feces and might be bitten during bat hunting. Thus, long-term systematic surveillance of bats is needed to determine the ecologic relationship between bats, humans, other animals, and the environment.

Recombination of Human Coxsackievirus B5 in Hand, Foot, and Mouth Disease Patients, China

Recombination of Human Coxsackievirus B5 in Hand, Foot, and Mouth Disease Patients, China

Outbreak of Porcine Epidemic Diarrhea in Suckling Piglets, China

Outbreak of Porcine Epidemic Diarrhea in Suckling Piglets, China

Novel Prion Protein in BSE-affected Cattle, Switzerland

Novel Prion Protein in BSE-affected Cattle, Switzerland

Schmallenberg Virus in Central Nervous System of Ruminants

Schmallenberg Virus in Central Nervous System of Ruminants

Iridovirus Infection in Chinese Giant Salamanders, China, 2010

Iridovirus Infection in Chinese Giant Salamanders, China, 2010