Synthetic transcripts of double-stranded Birnavirus genome are infectious.

We have developed a system for generation of infectious bursal disease virus (IBDV), a segmented double-stranded RNA virus of the Birnaviridae family, with the use of synthetic transcripts derived from cloned cDNA. Independent full-length cDNA clones were constructed that contained the entire coding and noncoding regions of RNA segments A and B of two distinguishable IBDV strains of serotype I. Segment A encodes all of the structural (VP2, VP4, and VP3) and nonstructural (VP5) proteins, whereas segment B encodes the RNA-dependent RNA polymerase (VP1). Synthetic RNAs of both segments were produced by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of Vero cells with combined plus-sense transcripts of both segments generated infectious virus as early as 36 hr after transfection. The infectivity and specificity of the recovered chimeric virus was ascertained by the appearance of cytopathic effect in chicken embryo cells, by immunofluorescence staining of infected Vero cells with rabbit anti-IBDV serum, and by nucleotide sequence analysis of the recovered virus, respectively. In addition, transfectant viruses containing genetically tagged sequences in either segment A or segment B of IBDV were generated to confirm the feasibility of this system. The development of a reverse genetics system for double-stranded RNA viruses will greatly facilitate studies of the regulation of viral gene expression, pathogenesis, and design of a new generation of live vaccines.

Density-dependent decline of host abundance resulting from a new infectious disease

Although many new diseases have emerged within the past 2 decades [Cohen, M. L. (1998) Brit. Med. Bull. 54, 523–532], attributing low numbers of animal hosts to the existence of even a new pathogen is problematic. This is because very rarely does one have data on host abundance before and after the epizootic as well as detailed descriptions of pathogen prevalence [Dobson, A. P. & Hudson, P. J. (1985) in Ecology of Infectious Diseases in Natural Populations, eds. Grenfell, B. T. & Dobson, A. P. (Cambridge Univ. Press, Cambridge, U.K.), pp. 52–89]. Month by month we tracked the spread of the epizootic of an apparently novel strain of a widespread poultry pathogen, Mycoplasma gallisepticum, through a previously unknown host, the house finch, whose abundance has been monitored over past decades. Here we are able to demonstrate a causal relationship between high disease prevalence and declining house finch abundance throughout the eastern half of North America because the epizootic reached different parts of the house finch range at different times. Three years after the epizootic arrived, house finch abundance stabilized at similar levels, although house finch abundance had been high and stable in some areas but low and rapidly increasing in others. This result, not previously documented in wild populations, is as expected from theory if transmission of the disease was density dependent.

Climate and infectious disease: Use of remote sensing for detection of Vibrio cholerae by indirect measurement

It has long been known that cholera outbreaks can be initiated when Vibrio cholerae, the bacterium that causes cholera, is present in drinking water in sufficient numbers to constitute an infective dose, if ingested by humans. Outbreaks associated with drinking or bathing in unpurified river or brackish water may directly or indirectly depend on such conditions as water temperature, nutrient concentration, and plankton production that may be favorable for growth and reproduction of the bacterium. Although these environmental parameters have routinely been measured by using water samples collected aboard research ships, the available data sets are sparse and infrequent. Furthermore, shipboard data acquisition is both expensive and time-consuming. Interpolation to regional scales can also be problematic. Although the bacterium, V. cholerae, cannot be sensed directly, remotely sensed data can be used to infer its presence. In the study reported here, satellite data were used to monitor the timing and spread of cholera. Public domain remote sensing data for the Bay of Bengal were compared directly with cholera case data collected in Bangladesh from 1992–1995. The remote sensing data included sea surface temperature and sea surface height. It was discovered that sea surface temperature shows an annual cycle similar to the cholera case data. Sea surface height may be an indicator of incursion of plankton-laden water inland, e.g., tidal rivers, because it was also found to be correlated with cholera outbreaks. The extensive studies accomplished during the past 25 years, confirming the hypothesis that V. cholerae is autochthonous to the aquatic environment and is a commensal of zooplankton, i.e., copepods, when combined with the findings of the satellite data analyses, provide strong evidence that cholera epidemics are climate-linked.

Epithelial antibiotic induced in states of disease

Epithelial defensins provide an active defense against the external microbial environment. We investigated the distribution and expression of this class of antimicrobial peptides in normal cattle and in animals in varying states of disease. β-defensin mRNA was found to be widely expressed in numerous exposed epithelia but was found at higher levels in tissues that are constantly exposed to and colonized by microorganisms. We observed induction in ileal mucosa during chronic infection with Mycobacterium paratuberculosis and in bronchial epithelium after acute infection with Pasteurella haemolytica. It has been proposed that expression of antimicrobial peptides is an integral component of the inflammatory response. The results reported here support this hypothesis and suggest that epithelial defensins provide a rapidly mobilized local defense against infectious organisms.

Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform

The scrapie prion protein (PrPSc) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrPC) by a conformational change. N-terminal truncation of PrPSc by limited proteolysis produces a protein of ≈142 residues designated PrP 27–30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27–30 was expressed in Escherichia coli and purified. After refolding rPrP into an α-helical form resembling PrPC, the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113–125 characterize a hydrophobic cluster, which packs against an irregular β-sheet, whereas residues 90–112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200–227) and a structured loop (residues 165–171) form a discontinuous epitope for binding of a protein that facilitates PrPSc formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrPC into PrPSc, whereas the discontinuous epitope near the C terminus controls this transition.

Neonatal gene transfer leads to widespread correction of pathology in a murine model of lysosomal storage disease

For many inborn errors of metabolism, early treatment is critical to prevent long-term developmental sequelae. We have used a gene-therapy approach to demonstrate this concept in a murine model of mucopolysaccharidosis type VII (MPS VII). Newborn MPS VII mice received a single intravenous injection with 5.4 × 106 infectious units of recombinant adeno-associated virus encoding the human β-glucuronidase (GUSB) cDNA. Therapeutic levels of GUSB expression were achieved by 1 week of age in liver, heart, lung, spleen, kidney, brain, and retina. GUSB expression persisted in most organs for the 16-week duration of the study at levels sufficient to either reduce or prevent completely lysosomal storage. Of particular significance, neurons, microglia, and meninges of the central nervous system were virtually cleared of disease. In addition, neonatal treatment of MPS VII mice provided access to the central nervous system via an intravenous route, avoiding a more invasive procedure later in life. These data suggest that gene transfer mediated by adeno-associated virus can achieve therapeutically relevant levels of enzyme very early in life and that the rapid growth and differentiation of tissues does not limit long-term expression.

Hepatitis C virus lacking the hypervariable region 1 of the second envelope protein is infectious and causes acute resolving or persistent infection in chimpanzees

Persistent infection with hepatitis C virus (HCV) is among the leading causes of chronic liver disease. Previous studies suggested that genetic variation in hypervariable region 1 (HVR1) of the second envelope protein, possibly in response to host immune pressure, influences the outcome of HCV infection. In the present study, a chimpanzee transfected intrahepatically with RNA transcripts of an infectious HCV clone (pCV-H77C) from which HVR1 was deleted became infected; the ΔHVR1 virus was subsequently transmitted to a second chimpanzee. Infection with ΔHVR1 virus resulted in persistent infection in the former chimpanzee and in acute resolving infection in the latter chimpanzee. Both chimpanzees developed hepatitis. The ΔHVR1 virus initially replicated to low titers, but virus titer increased significantly after mutations appeared in the viral genome. Thus, wild-type HCV without HVR1 was apparently attenuated, suggesting a functional role of HVR1. However, our data indicate that HVR1 is not essential for the viability of HCV, the resolution of infection, or the progression to chronicity.

Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein

Transmissible spongiform encephalopathies, or prion diseases, are fatal degenerative disorders of the central nervous system that affect humans and animals. Prions are nonconventional infectious agents whose replication depends on the host prion protein (PrP). Transmission of prions to cultured cells has proved to be a particularly difficult task, and with a few exceptions, their experimental propagation relies on inoculation to laboratory animals. Here, we report on the development of a permanent cell line supporting propagation of natural sheep scrapie. This model was obtained by stable expression of a tetracycline-regulatable ovine PrP gene in a rabbit epithelial cell line. After exposure to scrapie agent, cultures were repeatedly found to accumulate high levels of abnormal PrP (PrPres). Cell extracts induced a scrapie-like disease in transgenic mice overexpressing ovine PrP. These cultures remained healthy and stably infected upon subpassaging. Such data show that (i) cultivated cells from a nonneuronal origin can efficiently replicate prions; and (ii) species barrier can be crossed ex vivo through the expression of a relevant PrP gene. This approach led to the ex vivo propagation of a natural transmissible spongiform encephalopathy agent (i.e., without previous experimental adaptation to rodents) and might be applied to human or bovine prions.

Altered lymphocyte responses and cytokine production in mice deficient in the X-linked lymphoproliferative disease gene SH2D1A/DSHP/SAP

We have introduced a targeted mutation in SH2D1A/DSHP/SAP, the gene responsible for the human genetic disorder X-linked lymphoproliferative disease (XLP). SLAM-associated protein (SAP)-deficient mice had normal lymphocyte development, but on challenge with infectious agents, recapitulated features of XLP. Infection of SAP− mice with lymphocyte choriomeningitis virus (LCMV) or Toxoplasma gondii was associated with increased T cell activation and IFN-γ production, as well as a reduction of Ig-secreting cells. Anti-CD3-stimulated splenocytes from uninfected SAP− mice produced increased IFN-γ and decreased IL-4, findings supported by decreased serum IgE levels in vivo. The Th1 skewing of these animals suggests that cytokine misregulation may contribute to phenotypes associated with mutation of SH2D1A/SAP.

Naturally processed peptides from two disease-resistance-associated HLA-DR13 alleles show related sequence motifs and the effects of the dimorphism at position 86 of the HLA-DR beta chain.

HLA-DR13 has been associated with resistance to two major infectious diseases of humans. To investigate the peptide binding specificity of two HLA-DR13 molecules and the effects of the Gly/Val dimorphism at position 86 of the HLA-DR beta chain on natural peptide ligands, these peptides were acid-eluted from immunoaffinity-purified HLA-DRB1*1301 and -DRB1*1302, molecules that differ only at this position. The eluted peptides were subjected to pool sequencing or individual peptide sequencing by tandem MS or Edman microsequencing. Sequences were obtained for 23 peptides from nine source proteins. Three pool sequences for each allele and the sequences of individual peptides were used to define binding motifs for each allele. Binding specificities varied only at the primary hydrophobic anchor residue, the differences being a preference for the aromatic amino acids Tyr and Phe in DRB1*1302 and a preference for Val in DRB1*1301. Synthetic analogues of the eluted peptides showed allele specificity in their binding to purified HLA-DR, and Ala-substituted peptides were used to identify the primary anchor residues for binding. The failure of some peptides eluted from DRB1*1302 (those that use aromatic amino acids as primary anchors) to bind to DRB1*1301 confirmed the different preferences for peptide anchor residues conferred by the Gly-->Val change at position 86. These data suggest a molecular basis for the differential associations of HLA-DRB1*1301 and DRB1*1302 with resistance to severe malaria and clearance of hepatitis B virus infection.

Viral particles are required for infection in neurodegenerative Creutzfeldt-Jakob disease.

Several models have been proposed for the infectious agents that cause human Creutzfeldt-Jakob disease (CJD) and sheep scrapie. Purified proteins and extracted nucleic acids are not infectious. To further identify the critical molecular components of the CJD agent, 120S infectious material with reduced prion protein (PrP) was treated with guanidine hydrochloride or SDS. Particulate and soluble components were then separated by centrifugation and molecularly characterized. Conditions that optimally solubilized residual PrP and/or nucleic acid-protein complexes were used to produce subfractions that were assayed for infectivity. All controls retained > 90% of the 120S titer (approximately 15% of that in total brain) but lost > 99.5% of their infectivity after heat-SDS treatment (unlike scrapie fractions enriched for PrP). Exposure to 1% SDS at 22 degrees C produced particulate nucleic acid-protein complexes that were almost devoid of host PrP. These sedimenting complexes were as infectious as the controls. In contrast, when such complexes were solubilized with 2.5 M guanidine hydrochloride, the infectious titer was reduced by > 99.5%. Sedimenting PrP aggregates with little nucleic acid and no detectable nucleic acid-binding proteins had negligible infectivity, as did soluble but multimeric forms of PrP. These data strongly implicate a classical viral structure, possibly with no intrinsic PrP, as the CJD infectious agent. CJD-specific protective nucleic acid-binding protein(s) have already been identified in 120S preparations, and preliminary subtraction studies have revealed several CJD-specific nucleic acids. Such viral candidates deserve more attention, as they may be of use in preventing iatrogenic CJD and in solving a fundamental mystery.