Pathogenesis of a North American hantavirus, Black Creek Canal virus, in experimentally infected Sigmodon hispidus.

This report describes the first detailed analysis of the replication, persistence, and excretion of a North American hantavirus in its natural rodent reservoir. Black Creek Canal virus was isolated from Sigmodon hispidus (cotton rat) shortly after the identification of a hantavirus pulmonary syndrome (HPS) case occurring in southern Florida. Six-week-old male cotton rats were inoculated subcutaneously with 1,000 tissue culture infectious doses. Viral complementary RNA (vcRNA) was quantified as a means of determining the site(s) of viral activity (transcription and replication). In the first few weeks post inoculation (pi), vcRNA was detectable in every tissue examined except blood. The quantities of vcRNA decreased over time, and by five months pi it could be detected only in the brain. In addition to using a quantitative polymerase chain reaction (QPCR) as a means of measuring viral replication/transcription, attempts were made to reisolate virus from all tissue samples taken. Virus could be isolated from every solid tissue examined, and the titers appeared to decrease over time, similar to the QPCR results. However, in contrast to the QPCR results, infectious virus was still routinely detectable at low levels in adrenal gland, liver, kidney, and testicle 150 days pi. Although results of testing for vcRNA in the blood were uniformly negative, infectious virus was detected at one week pi, reached highest titers at two weeks, and decreased dramatically by three weeks. After three weeks pi, infectious virus could only be detected sporadically in blood. Virus was isolated from urine collected during the first 70 days pi and throughout the entire study period in feces and wet bedding. These data indicate that the viral infection can be separated into an acute phase associated with high virus titers, and a chronic or persistent phase associated with lower virus titers and continued shedding of virus in excreta.

Amino acid changes in the L polymerase protein of vesicular stomatitis virus which confer aberrant polyadenylation and temperature-sensitive phenotypes.

TsG16(I) is a temperature-sensitive mutant of vesicular stomatitis virus. In vitro, at the permissive temperature (31 degrees), it makes long poly(A) tracts, shows a larger increase in polyadenylation in the presence of S-adenosyl-homocysteine than its parental wt(Glasgow) virus, and makes an excess of polycistronic mRNA. In vitro transcription is also more thermosensitive than that of wt virus. Previous work suggested that there are at least two mutations in the L gene of tsG16(I), one effecting the poly(A)-associated phenotypes, the polycistronic phenotype, and the ability to grow at 34.7 degrees, the other affecting in vitro thermosensitivity for transcription and ability to grow at 37 degrees. We report further characterization of two revertants: 35G16p25, which grows at 34.7 degrees and has regained the wt poly(A), SAH and polycistronic RNA phenotypes; and 37G16p25, isolated from 35G16p25 based on growth at 37 degrees, which has regained the wt phenotype for in vitro thermosensitivity of transcription. Both revertants were shown to be due to intracistronic reversion[s] in the L gene. Sequencing of the L genes indicated that the tsG16(I) poly(A), SAH, polycistronic RNA, and growth at 34.7 degrees phenotypes were associated with amino acid 1488 phenylalanine-->serine and that transcription thermosensitivity and growth at 37 degrees were associated with changes in cysteine 1291.

The L protein of vesicular stomatitis virus modulates the response of the polyadenylic acid polymerase to S-adenosylhomocysteine.

TsG16(I) is a temperature-sensitive (ts) mutant of vesicular stomatitis virus, Indiana serotype, which overproduces polyadenylic acid [poly(A)] in an in vitro transcription system due to a mutation in the L protein. Others have reported that L-S-adenosylhomocysteine (S-Ado-Hcy) causes wild-type (wt) virus to overproduce poly(A) in vitro. The possibility that tsG16(I) constitutively expresses a property induced by S-Ado-Hcy in the case of wt virus was found not to be so since polyadenylation by the mutant was still sensitive to S-Ado-Hcy. Indeed, S-Ado-Hcy caused tsG16(I) to overproduce poly(A) in vitro to a greater extent than its parental wt virus. The increase in polyadenylation observed in response to saturating levels of S-Ado-Hcy differed for tsG16(I), for its parental wt virus and for another wt strain. To characterize which viral protein modulated the polyadenylation response to S-Ado-Hcy, purified virions were fractionated and their phenotypes in homologous and heterologous reconstitution assays were examined. The results indicated that the viral L protein modulated the response in all three stocks of virus. These data provide further evidence to suggest that the L protein of vesicular stomatitis virus plays a role in polyadenylation of the viral mRNA.

Sin Nombre virus mRNA synthesis.

Sin Nombre (SN) virus is the major etiologic agent of hantavirus pulmonary syndrome, a severe respiratory disease with high mortality. Like other hantaviruses, SN virus causes an inapparent chronic infection of the natural rodent reservoir and tends to grow slowly and produce little cytopathic effect even in highly susceptible Vero E6 tissue culture cells. An electrochemiluminescent quantitative PCR approach was developed to allow examination of SN virus RNA transcription in synchronously infected cells. Although virion particles contain equimolar ratios of the three negative-strand genome RNA segments (S, M, and L), rates and levels of accumulation of the corresponding N, GPC, and L viral mRNAs varied. The smallest mRNA (N) was detectable earliest and plateaued at the highest level, where as the largest mRNA (L) appeared latest and at the lowest plateau level. In addition, all three mRNAs were found to share a common 5' capped primer initiation mechanism, but appeared to have different mRNA termination mechanisms. The N mRNA 3' terminus mapped to position 1435 on the S segment, in close proximity to a CCC-rich suspected transcription termination motif. The GPC mRNA 3' terminus contained a poly(A) tall and mapped to a U8 transcription termination-polyadenylation motif reminiscent of those seen in other negative-strand RNA viruses. Finally, the L mRNA 3' terminus appeared identical to the L segment antigenome 3' terminus.

Increased synthesis of polycistronic mRNA associated with increased polyadenylation by vesicular stomatitis virus.

Electron microscopy suggested that the mRNA produced in vitro by tsG16(I), a temperature-sensitive mutant of vesicular stomatitis virus, contained an increased proportion of polycistronic mRNAs. Using hybrid selection, we found that the poly(A)+ mRNA synthesized in vitro by tsG16(I) contained approximately two to three times more polycistronic mRNA than did poly(A)+ mRNA synthesized in vitro by the parental wild-type (wt) virus. The increase in polycistronic mRNA occurred at all intergenic junctions examined. In vitro, tsG16(I) has an increased polyadenylation phenotype and a temperature-sensitive transcriptase activity that appear to be due to different mutations. Partial revertants of tsG16(I), which have lost the aberrant polyadenylation phenotype but retain the in vitro thermosensitive transcriptase, produced wt amounts of polycistronic mRNA. This suggested that the increased production of polycistronic mRNA by tsG16(I) may be associated with the increased polyadenylation phenotype of this mutant. These data further support the hypothesis that an increase in size of poly(A) tracts is associated with increased production of polycistronic mRNA.

Revertants of a mutant of vesicular stomatitis virus which has an aberrant polyadenylation activity and a temperature-sensitive transcriptase.

tsG16(l), a temperature-sensitive mutant of vesicular stomatitis virus, in vitro has at least three phenotypic differences from its parental wild-type (wt) virus due to mutation of the L gene. It was not known whether (i) the temperature-sensitivity of the transcriptase, (ii) the aberrant polyadenylation phenotype, and (iii) the extent of increased polyadenylation in response to S-adenosylhomocysteine (SAH) were associated with a single mutation. Spontaneous partial revertants were selected from tsG16(I) on the basis of the ability to form plaques at 34.7 degrees (35G16 revertants) or from 35G16 revertants on the basis of the ability to form plaques at 37 degrees (37G16 revertants). All six 35G16 revertants had fully (five) or partially (one) recovered the wt polyadenylation phenotype and the former five had also fully recovered the wt polyadenylation response to SAH. This suggested that a single mutation in tsG16(I) was probably associated with both of these phenotypes and also probably conferred the inability to grow at 34.7 degrees. None of the 35G16 revertants regained the wt phenotype for thermosensitivity of the transcriptase, although both of the 37G16 revertants did. This suggested that in vitro temperature-sensitivity of transcription by tsG16(I) might be due to a mutation different than the one affecting polyadenylation in the absence or presence of SAH.

Multiplex analysis of cytokines in the blood of cynomolgus macaques naturally infected with Ebola virus (Reston serotype).

Ebola virus (EBO) causes the most severe form of viral hemorrhagic fever in humans and nonhuman primates with up to 90% of infections culminating in death. The requirement of maximum containment laboratories for Ebola virus research has limited opportunities to study the pathogenesis of EBO infections. While tissue damage does occur, often it would appear not to be sufficient to explain death, indicating that soluble mediators play an important role in disease progression. In previous studies, fatal human infections with the Zaire subtype of Ebola (EBO-Z) were associated with an increase in the levels of inflammatory cytokines. In this investigation, a new multiplex assay was developed and used to measure circulating levels of cytokines and chemokines in cynomolgus macaques infected with the Reston subtype of EBO (EBO-R). Increased levels of IL-6, TNF-alpha, IFN-gamma, IL-2, IL-4, IL-8, IL-10, and GM-CSF were detected in infected animals, and the increase in circulating cytokines correlated with an increase in circulating viral antigen. Blood samples from animals showing high levels of cytokines were also tested for the chemokines: MCP-1, IL-1beta, MIP-1alpha, MIP-1beta, IP-10, and RANTES. High levels of MCP-1 and MIP-1beta, and RANTES were found in infected primates and, while levels were more variable, IL-1beta was detected only in infected animals.

Transmission of Black Creek Canal virus between cotton rats.

Black Creek Canal (BCC) virus is a hantavirus associated with hantavirus pulmonary syndrome in southeastern North America. The virus was isolated from the spleen of a cotton rat (Sigmodon hispidus) trapped in southern Florida. Our previous studies have shown that we could consistently infect male cotton rats with BCC virus in the laboratory. These animals became persistently infected and virus could be detected in salivary glands, urine, and feces. In this report we show: (1) female and male cotton rats are equally susceptible to BCC virus infection, (2) susceptibility to infection was not influenced by age, (3) all inoculated rats transmitted the infection to uninoculated cage mates, and (4) offspring of infected rats became infected despite the presence of high maternal antibodies. The course of BCC virus infection, as determined by antibody response and the ability to isolate or detect virus, appeared to be similar regardless of whether the rats obtained their infection by inoculation or contact with inoculated rats. J. Med. Virol. 60:70-76, 2000. Published 2000 Wiley-Liss, Inc.

Problem-based learning in a health sciences librarianship course.

Problem-based learning (PBL) has been adopted by many medical schools in North America. Because problem solving, information seeking, and lifelong learning skills are central to the PBL curriculum, health sciences librarians have been actively involved in the PBL process at these medical schools. The introduction of PBL in a library and information science curriculum may be appropriate to consider at this time. PBL techniques have been incorporated into a health sciences librarianship course at the School of Library and Information Science (LIS) at the University of Wisconsin-Milwaukee to explore the use of this method in an advanced Library and Information Science course. After completion of the course, the use of PBL has been evaluated by the students and the instructor. The modified PBL course design is presented and the perceptions of the students and the instructor are discussed.