- Volume 79, Issue 10, 1998
Volume 79, Issue 10, 1998
- Articles
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Promoter activity associated with the intergenic regions of banana bunchy top virus DNA-1 to −6 in transgenic tobacco and banana cells
More LessPromoter regions associated with each of the six ssDNA components of banana bunchy top virus (BBTV) have been characterized. DNA segments incorporating the intergenic regions of BBTV DNA-1 to -6 were isolated and fused to the uidA (β-glucuronidase) reporter gene to assess promoter activity. In tobacco cell suspensions, the BBTV DNA- 2 and -6 promoters generated levels of GUS expression 2-fold greater and similar to the 800 bp CaMV 35S promoter, respectively. Deletion analysis of the BBTV DNA-6 promoter suggested all the necessary promoter elements required for strong expression were located within 239 nucleotides upstream of the translational start codon. In transgenic tobacco plants, the BBTV-derived promoters generally provided a weak, tissue-specific GUS expression pattern restricted to phloem-associated cells. However, in callus derived from tobacco leaf tissue, GUS expression directed by the BBTV DNA-6 promoter was strong and, in some lines, comparable to the CaMV 35S promoter. Detectable promoter activity associated with the BBTV promoters in banana embryogenic cells was only observed using a sensitive green fluorescent protein (GFP) reporter. Promoters derived from BBTV DNA-4 and -5 generated the highest levels of transient activity, which were greater than that of the maize ubi-1 promoter. In transgenic banana plants, the activity of the BBTV DNA-6 promoter was restricted to the phloem of leaves and roots, stomata and root meristems.
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Molecular characterization of a new whitefly-transmissible bipartite geminivirus infecting tomato in Panama
More LessThe nucleotide sequence of infectious clones of a tomato-infecting geminivirus from Panama [named tomato leaf curl virus (ToLCV-Pan) because of symptoms produced in infected tomato (plant stunting and mild leaf curling)] was determined. ToLCV-Pan has a bipartite genome (DNAs A and B) and computer analysis showed that the genome resembles that of other bipartite, whitefly-trans- mitted geminiviruses. DNA A (2584 nt) and B (2542 nt) have little sequence homology other than within the common region. ToLCV-Pan clones were introduced into Lycopersicon esculentum and infected plants developed the same symptoms as naturally infected tomatoes. Homology analysis of DNA A and B showed that ToLCV-Pan is most closely related to potato yellow mosaic virus (PYMV) from Venezuela. Pseudorecombination between ToLCV-Pan and PYMV did not give viable pseudorecombinant viruses. However, in some plants infected with the pseudorecombinant virus produced by ToLCV-Pan DNA A and PYMV DNA B, systemic movement of ToLCV-Pan DNA A was observed.
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Localization of the P1 protein of potato Y potyvirus in association with cytoplasmic inclusion bodies and in the cytoplasm of infected cells
More LessThe N-terminal P1 proteinase of potato virus Y (ordinary strain group isolate PVY-O) was expressed in E. coli. Antiserum was raised against the expressed protein and used to detect the viral proteins in infected tobacco leaf tissue by Western blotting and by electron microscopy with immuno- gold labelling. In the immunogold localization studies P1 protein was detected in association with the cytoplasmic inclusion bodies characteristic of PVY infections and in the cytoplasm of the infected plant cells. No significant P1 antibody binding with other plant cell organelles, or with the cell wall and plasmodesmata, was detected by immunogold labelling.
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Specific inclusion bodies are associated with replication of lettuce infectious yellows virus RNAs in Nicotiana benthamiana protoplasts
More LessNicotiana benthamiana mesophyll protoplasts, either mock-inoculated or inoculated using in vitro transcripts derived from lettuce infectious yellows virus (LIYV) RNA 1- and/or RNA 2-cloned cDNAs were analysed by transmission electron microscopy (TEM) and, in some cases, also by immunogold labelling. TEM revealed the main cytopathological effects of LIYV infections in N. benthamiana protoplasts infected with RNAs 1 and 2: (a) typical closterovirus-induced (beet yellows virus-type) accumulations of vesiculated cytoplasmic membranes as inclusion bodies, sometimes with associated virions; (b) scattered aggregations of virions within the cytoplasm; and (c) electron-dense plasmalemma deposits. These were not seen in mock-inoculated protoplasts. Protoplasts inoculated only with LIYV RNA 1 contained vesiculated cytoplasmic inclusion bodies, but not virions or plasmalemma deposits. Thus, infection by only LIYV RNA 1 is sufficient to induce characteristic clostero- virus vesiculated cytoplasmic inclusion bodies. However, both LIYV RNAs 1 and 2 are needed for production of virions and plasmalemma deposits.
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Diversity among isolates of squash mosaic virus
More LesscDNA clones of RNA-2 of two isolates of squash mosaic virus (SqMV) were constructed and sequenced, revealing 87 % sequence similarity. In Northern blot hybridization analyses, DNA probes made from these clones defined two SqMV hybridization subgroups. This grouping was verified by reciprocal hybridizations of purified RNA from five SqMV isolates, as probed with cDNA made from a member of each subgroup. Comparison of the RNA-2 sequence among the two SqMV isolates, and the reported sequence of other comoviruses, showed that SqMV constitutes one of four major branches in a phylogenetic tree of the genus. Analysis of the terminal noncoding sequences showed that although potentially similar folding patterns may form, neither nucleotide sequence nor secondary structural elements are highly conserved among comoviruses. In vitro translation products from purified RNA-1 of each subgroup (encoding the viral proteases) were found to process the polyprotein generated by in vitro translation of purified RNA-2 from either subgroup.
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Association of sequences in the coat protein/readthrough domain of potato mop-top virus with transmission by Spongospora subterranea
More LessA monofungal culture of Spongospora subterranea was unable to acquire and transmit the T isolate of potato mop-top pomovirus (PMTV-T), which has been maintained by manual transmission in the laboratory for 30 years. A recently obtained field isolate (PMTV-S) was efficiently acquired and transmitted by the same fungus culture. Sequence analysis of the readthrough (RT) protein-coding region of PMTV-S showed the presence of an additional 543 nt in the 3′ half of the coding region relative to that of PMTV-T. These additional nucleotides preserved the reading frame of the RT protein and inserted 181 amino acids into the RT protein. This was confirmed by a comparison by immuno- blotting of the sizes of the RT protein of PMTV-T and other recent isolates of PMTV.
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Nucleotide sequence, genetic organization and expression strategy of the double-stranded RNA associated with the ‘447’ cytoplasmic male sterility trait in Vicia faba
More LessThe entire nucleotide sequence of the doublestranded (ds) RNA associated with the unconventional ‘ 447 ’ cytoplasmic male sterility (CMS) trait in Vicia faba was determined from overlapping cDNA clones and by RT-PCR. Confirming previous observations, it was found that the negative-strand was continuous and 17 635 nt long, while the positive- strand featured an interruption, probably a nick, that could potentially define two subgenomic RNAs of 2735 nt and 14900 nt, with the smaller RNA being located on the 5′ side. The entire positive- strand could encode a single in-frame ORF starting at the first AUG at position 42–44 and ending with a TGA at 17 517–17 519. This long potential polypeptide with a predicted molecular mass of 654109 is the largest described to date in the plant kingdom and contains conserved amino acid sequence motifs typical of viral helicases and RNA-dependent RNA polymerases (RDRP). Only limited sequence homology was detected with the ORF B encoded by the hypovirulence-associated dsRNA of chestnut blight fungus, a dsRNA replicon similarly contained in host-derived membranous vesicles and considered to share a common ancestry with potyviruses. By contrast, the helicase and RDRP domains were in the same respective arrangement and shared extensive sequence homologies with those identified in the polyprotein encoded by the dsRNA isolated from Japonica rice, another dsRNA replicon featuring a specific nick in the positive-strand. Although no proteolytic self-cleavage activity has yet been demonstrated, it appears likely that this long ORF is a polyprotein that undergoes proteolytic maturation, with one of the polypeptides derived by selfcleavage being the determinant of the CMS trait.
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Translation efficiencies of the 5′ untranslated region from representatives of the six major genotypes of hepatitis C virus using a novel bicistronic reporter assay system
More LessThe 5′ untranslated region (5′UTR) of hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) which directs translation of the viral open reading frame (ORF). The 5′UTR is highly conserved between virus isolates in both primary sequence and predicted secondary structure. We cloned and sequenced the 5′ regions (nt 18 of the 5′UTRto nt 15 of the core coding sequence) of HCV isolates representing the six major genotypes and subcloned these into a bicistronic, dual luciferase reporter construct. The relative expression of the two luciferases, one directed by the HCV IRES and the other by cap-dependent ribosome scanning, was used to compare the activities of the different IRES elements in transfected cells. The 5′UTR from a genotype 2b isolate was the most efficient at directing translation in all four cell lines tested: BHK-21, HeLa-T4 , HuH7 and HepG2. In HepG2 cells the 2b 5′UTR was three times as efficient as the type 6a 5′UTR, which was generally the least active IRES tested. These data suggest that HCV isolates are not able to translate their ORF with equal efficiency, and provide a starting point from which further sequence-function studies can be undertaken.
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In vitro infection of adult normal human hepatocytes in primary culture by hepatitis C virus
In vitro infection of adult normal human hepatocytes in primary culture has been performed for investigating the replication cycle of hepatitis C virus (HCV) in differentiated cells. Hepatocytes were prepared from liver tissue resected from donors who tested negative for HCV, and inoculation was performed 3 days after plating with 33 HCV serum samples of different virus load and genotype. The presence of intracellular HCV RNA, detected by a strand-specific rTth RT-PCR assay, was used as evidence of infection. A kinetics analysis of HCV replication revealed that intracellular negative-strand RNA appeared at day 1 post-infection with a maximum level at days 3 and 5, followed by a decrease until day 14. At day 5, we estimated that the copy level of viral RNA was amplified at least 15-fold in infected cells. The level of intracellular HCV RNA in response to different serum samples was reproducible from one hepatocyte culture to another, suggesting that there is no inter-individual variability in the susceptibility of hepatocytes to HCV infection. These findings indicate that adult human hepatocytes in primary culture retain their susceptibility to in vitro HCV infection and support HCV RNA replication. This model should represent a valuable tool for the study of initial steps of the HCV replication cycle and for the evaluation of antiviral molecules.
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West Nile virus envelope proteins: nucleotide sequence analysis of strains differing in mouse neuroinvasiveness
More LessSeveral neuroinvasive and non-neuroinvasive West Nile (WN) viruses were characterized by nucleotide sequencing of their envelope (E) protein regions. Prolonged passage in mosquito cells caused loss of neuroinvasiveness and acquisition of an N-linked glycosylation site, which is utilized. Limited passage in cell culture also caused glycosylation but not attenuation, suggesting that glycosylation may not be directly responsible for attenuation and that a second mutation (L68 → P) may also be involved. A monoclonal antibody-neutralization escape mutant with a substitution at residue 307, a site common to other flavivirus escape mutants, was also attenuated. A partially neuroinvasive revertant regained the parental E sequence, implying that determinants outside of the E region may also influence attenuation. Data suggest that the neuroinvasive determinants may be similar to those for other flaviviruses. Also, sequence comparison with the WN virus (Nigeria) strain revealed considerable divergence of the E protein at the nucleotide and amino acid levels.
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The antiviral compound ribavirin modulates the T helper (Th) 1/Th2 subset balance in hepatitis B and C virus-specific immune responses
More LessRibavirin is effective in combination therapies against chronic hepatitis C virus (HCV) infection, although its direct antiviral properties are unclear. We therefore studied the immune-modulatory effects of ribavirin on hepatitis B virus (HBV)- and HCV-specific immune responses. During a 24 week placebo-controlled ribavirin trial in ten patients with chronic HCV infection, HCV antibodies and alanine aminotransferase (ALT) levels decreased transiently whereas the serum levels of HCV RNA remained stable. Effects of ribavirin on human and murine phytohaemagglutinin (PHA)-activated T cells included inhibition of in vitro proliferation and modulation of IL-2, IL-4, IFN-γ and TNF-α levels. HBcAg- and HBeAg-specific IL-2 and IFN-γ levels were ≥ 25-fold higher in mice immunized with HBV core- or e-antigens (HBcAg, HBeAg) while receiving riba virin compared to untreated mice, but IL-4 and IL-6 remained constant. Concordantly, a slight shift was observed in the IgG subclass distribution of the humoral responses of ribavirin-treated mice to HBeAg and HCV NS3 protein. Ribavirin treatment of HBeAg-transgenic (HBeAg-Tg) mice induced a dose- dependent down-regulation of T helper (Th)2- mediated antibody production to HBeAg. In ribavirin-treated HBeAg-Tg mice anti-HBe IgG1 (positively regulated by Th2 cytokines) decreased simultaneously as both anti-HBe IgG2a (positively regulated byTh1 cytokines) levels and in vitro T-cell IFN-γ production increased, indicating a change in the Th1/Th2 balance. Thus, the present data suggest that ribavirin is not strictly an antiviral compound, but rather it alters the T-cell balance in the immune system.
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Cell-to-cell spread of poliovirus in the spinal cord of bonnet monkeys (Macaca radiata)
More LessIn order to study the spread of poliovirus in the spinal cord of bonnet monkeys, 108 TCID50 Mahoney strain of poliovirus was inoculated into the ulnar nerves of monkeys that were subsequently autop- sied on days 1,2, 3, 6, 9, 12, 14, 15 and 16 postinoculation (p.i.). Virus spread in the spinal cord, the accompanying histopathological changes and paralysis occurred in a cervico-thoraco-lumbar direction. Virus reached the cervical region of the spinal cord within the first 3 days and subsequently spread to all segments of the spinal cord. In situ hybridization demonstrated viral RNA initially in the cervical neurons on day 3 p.i. and in the anterior horn neurons of lumbar segments of the spinal cord by day 6 p.i. Loss of Nissl substance in some of the anterior horn neurons was apparent on day 3 p.i. in the cervical and thoracic regions and by day 6 p.i. in the lumbar region. In the lumbar region, neuro- nophagia was a consistent feature which was observed on days 6–9 p.i., followed by neuronal dropouts on day 12 p.i. and thereafter. In the cervical and thoracic region, reappearance of Nissl substance was apparent from day 12 p.i. Upper limb paralysis preceded lower limb paralysis (5·5 1·73 vs 8·18 2·18, P= 0·046), further suggesting that virus spread within the spinal cord was via an intraneural route despite persistent viraemia detectable from day 2 p.i. onwards. The temporal distribution of the virus spread, distribution of viral RNA, histopathological and clinical changes indicate a cell-to-cell spread of poliovirus in the CNS, having gained access to the CNS from the peripheral nerve.
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The Semliki Forest virus vector induces p53-independent apoptosis
Three deletion mutants of the structural protein region of the Semliki Forest virus (SFV) genome, including one which encompassed all the viral structural protein genes, induced apoptosis in BHK cells at 48 h after transfection, as shown by DNA laddering and TUNEL staining, as did the wild-type SFV4 RNA. A similar result was obtained for the SFV1 expression vector, which has a multicloning site inserted in place of the structural protein genes. However, in cells transfected with viral RNA containing a deletion of the nsP2 gene, neither viral RNA synthesis nor the induction of apoptosis occurred. Both SFV1 vector and wild-type SFV4 RNA induced apoptosis in human H358a lung carcinoma cells, which have a homozygous deletion of the p53 gene. It is concluded that the SFV vector encodes a function in the nonstructural coding region which induces p53-independent apoptosis and is dependent on viral RNA synthesis.
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Pathogenesis of pneumovirus infections in mice: detection of pneumonia virus of mice and human respiratory syncytial virus mRNA in lungs of infected mice by in situ hybridization.
More LessThe pathogenesis of pneumonia virus of mice (PVM) and human respiratory syncytial virus (HRSV) in BALB/c mice were investigated by using in situ hybridization to detect virus mRNA in fixed lung sections. Following intranasal inoculation with 120 p.f.u. PVM the pattern of hybridization showed that virus mRNA was initially detected within 2 days in alveolar cells. As the infection progressed the number of hybridizing alveolar cells increased and signal was also detected in cells lining the terminal bronchioles. By days 4 to 5 post-infection areas of morphological abnormality could be seen, particularly in the strongly hybridizing regions of the lung, and this correlated with the appearance of clinical signs of infection. In animals which survived the infection virus-specific mRNA could not be detected 10 days post-infection. Mice infected with 1500 p.f.u. HRSV showed significant differences in the distribution of virus-specific mRNA when compared to the pattern seen with PVM. HRSV mRNA was detected over large areas, but predominantly in peribronchiolar and perivascular regions of the lungs 5 days post-infection. The yield of PVM from infected mouse lungs was considerably higher than that of HRSV. The possible implications of these results for the use of the mouse model for pneumovirus infections are discussed.
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Nucleotide sequences of the trailer, nucleocapsid protein gene and intergenic regions of Newcastle disease virus strain Beaudette C and completion of the entire genome sequence.
More LessThe nucleotide sequences of the nucleocapsid protein (NP) gene, the intergenic regions in the nucleocapsid protein (NP)-phosphoprotein (P), P-matrix protein (M) and M-fusion glycoprotein gene junctions and the trailer region of a virulent Newcastle disease virus (NDV) strain Beaudette C were determined. The NP gene is 1747 nt long and encodes a protein of 489 amino acids. Each of the intergenic sequences determined is 1 nt long and, including the previously published intergenic sequences, the gene junction sequences varied in length from 1– 47 nt and lacked any sequence identity. The 5′ trailer region is 113 nt in length. Comparison of the sequences of the terminal leader and trailer regions of Beaudette C strain with those of nonvirulent strain B1 showed a high level of conservation, indicating the likelihood of these elements not being a factor in virulence. Together with previously published data, this report completes the sequence of the 15 186 nt genomic RNA of NDV strain Beaudette C.
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M protein correlates with the receptor-binding specificity of haemagglutinin protein of reassortant influenza A (H1N1) virus.
More LessFrom the reassortment experiments between A/ Aichi/4/92 and A/WSN/33 (WSN) (H1N1) viruses, two different phenotype viruses which contained the haemagglutinin (HA) gene from A/Aichi/4/92 virus and the neuraminidase (NA) gene from WSN virus were obtained. PW13 and PW15 viruses agglutinated chicken red blood cells (CRBC), while PW10 and PW70 viruses did not. However, the expressed HA proteins of these viruses did not adsorb CRBC. The difference in gene constellation between PW13, PW15 and PW10, PW70 viruses was the membrane protein (M) gene. The former two had the M gene from A/Aichi/4/92 virus and the latter two had that from WSN virus. In PW15- infected cells, haemadsorption of CRBC was observed 30 min later than that of goose red blood cells and the M1 protein migrated from the nucleus to the cytoplasm 30 min earlier than adsorption of CRBC was observed. On the other hand, in PW10- infected cells, haemadsorption of CRBC was not observed through the virus replication and the M1 protein stayed in the nucleus after HA and NA activities reached maximum levels. Co-expression of the M and the HA proteins of A/Aichi/4/92 virus did not help the HA protein gain the ability to adsorb CRBC. However, neuraminidase treatment of COS cells expressing the HA protein of A/Aichi/4/92 virus or MDCK cells infected by PW10 virus restored the ability to adsorb CRBC. We discussed the possibility that the M1 protein helped the NA protein in its role to modify the HA protein on the cell surface.
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The M1 and NP proteins of influenza A virus form homo- but not heterooligomeric complexes when coexpressed in BHK-21 cells.
More LessThe nucleoprotein (NP) and matrix protein (M1) are the most abundant structural proteins of influenza A virus. M1 forms a protein layer beneath the viral envelope and NP constitutes the protein backbone of the ribonucleoproteins (RNPs). In order to elucidate the functions of these proteins in virus assembly we have expressed NP and M1 in BHK-21 cells using Semliki Forest virus replicons and analysed their molecular interactions. We found that both M1 and NP engaged in extensive homooligomerization reactions soon after synthesis. However, there was no detectable heterooligomerization taking place between the two viral proteins, nor between these and host proteins. One interpreta-tion of these results is that homooligomers, and not monomers, of NP and M1 are used as building blocks during RNP assembly and formation of the submembranous M1 layer, respectively. The complete absence of M1-NP heterooligomers suggests, on the other hand, that these two major viral proteins do not interact directly with each other during virus assembly. We also found that a fraction of M1 associated with cellular membranes. This did not, however, result in membrane budding or vesicularization as was the case with the matrix protein of vesicular stomatitis virus when expressed separately (P. A. Justice and others, Journal of Virology 69, 3156– 3160, 1995).
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Enhancement of human immunodeficiency virus type 1 infectivity by Nef is producer cell-dependent.
The growth kinetics of wild-type and nef mutant viruses of human immunodeficiency virus type 1 were comparatively analysed in several human CD4 cell lines. Delayed replication of nef mutant virus was observed in all cell lines examined. To determine the stage in the virus replication cycle that is affected by Nef, a single-round replication assay was performed. Initially, the expression of marker genes in transfected cells was examined in order to study the role of Nef in the late phase of infection. The results obtained indicated that Nef is dispensable during the transcription to virion pro duction stage. Next, the effect of Nef on the early phase was investigated with a single-round infection. It was demonstrated that Nef is required in the early phase of the virus replication cycle, from virion adsorption to integration. Finally, the infectivity of virus stocks prepared from four cell lines was determined. The relative infectivity of the nef mutant from the four cell lines differed. Taken together, we conclude that Nef acts via modulation of viral particles to enhance virus infectivity in a cell- dependent manner.
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Human immunodeficiency virus type 1 in faeces and serum: evidence against independently evolving subpopulations.
More LessIt is not known whether independent tissue-specific evolution accounts for the differences between human immunodeficiency virus type 1 (HIV-1) subpopulations in intestinal tissue and blood. To study this, sequential serum samples from three persons were analysed for the presence of HIV-1 V3 genotypes which were detected exclusively in faeces at a specific time-point. For two persons the faeces genotype was found in serum samples collected before the time of faeces collection: 7 months for one person and 32 months for the other person. In the third person, serum collected 1 month after faeces collection contained the faeces genotype in abundance. These data indicate that a difference between intestinal tissue and blood HIV-1 subpopulations is not the result of complete compart- mentalization and independent HIV-1 evolution in intestinal tissue, but that it reflects an unequal distribution of HIV-1 in different tissues.
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Determinants of disease in the simian immunodeficiency virus-infected rhesus macaque: characterizing animals with low antibody responses and rapid progression.
Clinical and laboratory markers of simian immunodeficiency virus (SIV) infection were studied during the first 3 months after intravenous inoculation of rhesus macaques. Virus-binding serum antibody titres were correlated strongly with disease progression (P < 0·005) and were predictive of disease outcome by 7 weeks after inoculation. Low virusbinding serum antibody responses to SIV occurred in animals that also showed acute depletion of circulating CD20 B cells. Acute damage to the CD4 T cell and CD20 B cell populations rendered some animals incapable of mounting virus-specific antibody responses and these macaques became the rapidly progressing cases comprising approximately 20– 30% of infected animal cohorts.
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