Biomedical subjects

Allen Grolla

Publications and source records attributed to Allen Grolla.

15 recordsLinked to original sources

PubMed2006

Rapid and simple detection of Ebola virus by reverse transcription-loop-mediated isothermal amplification.

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and nonhuman primates with high mortality rates. Rapid identification of the virus is required to prevent spread of the infection. In this study, we developed and evaluated a one-step simple reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for the rapid detection of Zaire ebolavirus (ZEBOV), the most virulent species of EBOV, targeting the trailer region of the viral genome. The assay could detect 20 copies of the artificial ZEBOV RNA in 26 min with a real time-monitoring detection, and also detect 10(-3) FFU of the cell-culture propagated viruses. The reaction time needed to detect 10(4) FFU of ZEBOV was only 20 min. In addition, the assay was highly specific for ZEBOV. The RT-LAMP assay developed in this study is rapid, simple, highly specific, and sensitive for the detection of ZEBOV, and so may be an effective diagnostic tool. Furthermore, as this technique does not require sophisticated instrumentation, it seems very suitable for diagnosis in the field or laboratories in Ebola outbreak areas such as Central Africa.

Animals↗

PubMed2006

Rapid molecular strategy for filovirus detection and characterization.

Filoviruses have the capacity to cause lethal outbreaks of hemorrhagic fever in primates. Here we present a simple consensus reverse transcription-PCR method for filovirus recognition and characterization and demonstrate its utility with all known filovirus strains. Phylogenetic assignment is achieved by automated web-based sequence analysis of amplification products.

Animals↗

PubMed2006

Postexposure protection against Marburg haemorrhagic fever with recombinant vesicular stomatitis virus vectors in non-human primates: an efficacy assessment.

BACKGROUND: Effective countermeasures are urgently needed to prevent and treat infections caused by highly pathogenic and biological threat agents such as Marburg virus (MARV). We aimed to test the efficacy of a replication-competent vaccine based on attenuated recombinant vesicular stomatitis virus (rVSV), as a postexposure treatment for MARV haemorrhagic fever. METHODS: We used a rhesus macaque model of MARV haemorrhagic fever that produced 100% lethality. We administered rVSV vectors expressing the MARV Musoke strain glycoprotein to five macaques 20-30 min after a high-dose lethal injection of homologous MARV. Three animals were MARV-positive controls and received non-specific rVSV vectors. We tested for viraemia, undertook analyses for haematology and serum biochemistry, and measured humoral and cellular immune responses. FINDINGS: All five rhesus monkeys that were treated with the rVSV MARV vectors as a postexposure treatment survived a high-dose lethal challenge of MARV for at least 80 days. None of these five animals developed clinical symptoms consistent with MARV haemorrhagic fever. All the control animals developed fulminant disease and succumbed to the MARV challenge by day 12. MARV disease in the controls was indicated by: high titres of MARV (10(3)-10(5) plaque-forming units per mL); development of leucocytosis with concurrent neutrophilia at end-stage disease; and possible damage to the liver, kidney, and pancreas. INTERPRETATION: Postexposure protection against MARV in non-human primates provides a paradigm for the treatment of MARV haemorrhagic fever. Indeed, these data suggest that rVSV-based filoviral vaccines might not only have potential as preventive vaccines, but also could be equally useful for postexposure treatment of filoviral infections.

Animals↗

PubMed2006

Detection of Ebola virus in oral fluid specimens during outbreaks of Ebola virus hemorrhagic fever in the Republic of Congo.

BACKGROUND: Patients who have refused to provide blood samples has meant that there have been significant delays in confirming outbreaks of Ebola virus hemorrhagic fever (EVHF). During the 2 EVHF outbreaks in the Republic of Congo in 2003, we assessed the use of oral fluid specimens versus serum samples for laboratory confirmation of cases of EVHF. METHODS: Serum and oral fluid specimens were obtained from 24 patients with suspected Ebola and 10 healthy control subjects. Specimens were analyzed for immunoglobulin G antibodies by enzyme-linked immunosorbent assay (ELISA) and for Ebola virus by antigen detection ELISA and reverse-transcriptase polymerase chain reaction (RT-PCR). Oral fluid specimens were collected with a commercially available collection device. RESULTS: We failed to detect antibodies against Ebola in the oral fluid specimens obtained from patients whose serum samples were seropositive. All patients with positive serum RT-PCR results also had positive results for their oral fluid specimens. CONCLUSIONS: This study demonstrates the usefulness of oral fluid samples for the investigation of Ebola outbreaks, but further development in antibodies and antigen detection in oral fluid specimens is needed before these samples are used for filovirus surveillance activities in Africa.

Adult↗

PubMed2006

Cross-protection against Marburg virus strains by using a live, attenuated recombinant vaccine.

Marburg virus (MARV) has been associated with sporadic episodes of hemorrhagic fever, including a recent highly publicized outbreak in Angola that produced severe disease and significant mortality in infected patients. MARV is also considered to have potential as a biological weapon. Recently, we reported the development of a promising attenuated, replication-competent vaccine against MARV based on recombinant vesicular stomatitis virus (VSV) expressing the glycoprotein of the Musoke strain of MARV (VSVDeltaG/MARVGP-Musoke). We used this vaccine to demonstrate complete protection of cynomolgus monkeys against a homologous MARV challenge. While these results are highly encouraging, an effective vaccine would need to confer protection against all relevant strains of MARV. Here, we evaluated the protective efficacy of the VSVDeltaG/MARVGP-Musoke vaccine against two heterologous MARV strains, the seemingly more pathogenic Angola strain and the more distantly related Ravn strain. In this study, seven cynomolgus monkeys were vaccinated with the VSVDeltaG/MARVGP-Musoke vector. Three of these animals were challenged with the Angola strain, three with the Ravn strain, and a single animal with the Musoke strain of MARV. Two animals served as controls and were each injected with a nonspecific VSV vector; these controls were challenged with the Angola and Ravn strains, respectively. Both controls succumbed to challenge by day 8. However, none of the specifically vaccinated animals showed any evidence of illness either from the vaccination or from the MARV challenges and all of these animals survived. These data suggest that the VSVDeltaG/MARVGP-Musoke vaccine should be sufficient to protect against all known MARV strains.

Animals↗

PubMed2006

MassTag polymerase chain reaction for differential diagnosis of viral hemorrhagic fever.

Viral hemorrhagic fevers are associated with high rates of illness and death. Although therapeutic options are limited, early differential diagnosis has implications for containment and may aid in clinical management. We describe a diagnostic system for rapid, multiplex polymerase chain reaction identification of 10 different causes of viral hemorrhagic fevers.

Diagnosis, Differential↗

PubMed2005

Development of a new vaccine for the prevention of Lassa fever.

BACKGROUND: Recent importation of Lassa fever into Germany, the Netherlands, the United Kingdom, and the United States by travelers on commercial airlines from Africa underscores the public health challenge of emerging viruses. Currently, there are no licensed vaccines for Lassa fever, and no experimental vaccine has completely protected nonhuman primates against a lethal challenge. METHODS AND FINDINGS: We developed a replication-competent vaccine against Lassa virus based on attenuated recombinant vesicular stomatitis virus vectors expressing the Lassa viral glycoprotein. A single intramuscular vaccination of the Lassa vaccine elicited a protective immune response in nonhuman primates against a lethal Lassa virus challenge. Vaccine shedding was not detected in the monkeys, and none of the animals developed fever or other symptoms of illness associated with vaccination. The Lassa vaccine induced strong humoral and cellular immune responses in the four vaccinated and challenged monkeys. Despite a transient Lassa viremia in vaccinated animals 7 d after challenge, the vaccinated animals showed no evidence of clinical disease. In contrast, the two control animals developed severe symptoms including rashes, facial edema, and elevated liver enzymes, and ultimately succumbed to the Lassa infection. CONCLUSION: Our data suggest that the Lassa vaccine candidate based on recombinant vesicular stomatitis virus is safe and highly efficacious in a relevant animal model that faithfully reproduces human disease.

Animals↗

PubMed2005

Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses.

Vaccines and therapies are urgently needed to address public health needs stemming from emerging pathogens and biological threat agents such as the filoviruses Ebola virus (EBOV) and Marburg virus (MARV). Here, we developed replication-competent vaccines against EBOV and MARV based on attenuated recombinant vesicular stomatitis virus vectors expressing either the EBOV glycoprotein or MARV glycoprotein. A single intramuscular injection of the EBOV or MARV vaccine elicited completely protective immune responses in nonhuman primates against lethal EBOV or MARV challenges. Notably, vaccine vector shedding was not detectable in the monkeys and none of the animals developed fever or other symptoms of illness associated with vaccination. The EBOV vaccine induced humoral and apparent cellular immune responses in all vaccinated monkeys, whereas the MARV vaccine induced a stronger humoral than cellular immune response. No evidence of EBOV or MARV replication was detected in any of the protected animals after challenge. Our data suggest that these vaccine candidates are safe and highly efficacious in a relevant animal model.

Animals↗

PubMed2005

Rapid, differential diagnosis of orthopox- and herpesviruses based upon real-time PCR product melting temperature and restriction enzyme analysis of amplicons.

Orthopoxviruses tend to have non-specific early symptoms that cannot be differentiated readily from other infectious exanthemas, such as varicella-zoster virus (VZV) or disseminated herpes simplex virus (HSV) infections. A rapid assay was developed for the differential diagnosis of orthopoxviruses and herpesviruses based upon the melting temperatures of real-time PCR amplicons. A mean melting temperature difference of 8.7 degrees C was observed between the products amplified from the two virus families. Further identification of individual pathogens was made using restriction enzyme analysis. The assay was able to identify correctly viruses from quality control panels of herpes and orthopoxviruses.

DNA Primers↗

PubMed2004

Sequence determination of the Crimean-Congo hemorrhagic fever virus L segment.

Crimean-Congo hemorrhagic fever (CCHF) virus is highly pathogenic for humans and remains the only Category A virus for which full sequence information is currently unavailable. In this study we completed CCHF genome characterization by determining the L segment sequence using Dugbe and CCHF virus-specific oligonucleotides. Sequence alignments revealed the presence of four previously described conserved regions in all Bunyaviridae polymerases. Interestingly, additional regions containing putative Ovarian Tumor (OTU)-like cysteine protease and helicase domains were identified in the L segments of CCHF and Dugbe viruses, suggesting an autoproteolytic cleavage process for nairovirus L proteins.

Amino Acid Sequence↗

PubMed2004

Interpretation of diagnostic laboratory tests for severe acute respiratory syndrome: the Toronto experience.

BACKGROUND: An outbreak of severe acute respiratory syndrome (SARS) began in Canada in February 2003. The initial diagnosis of SARS was based on clinical and epidemiological criteria. During the outbreak, molecular and serologic tests for the SARS-associated coronavirus (SARS-CoV) became available. However, without a "gold standard," it was impossible to determine the usefulness of these tests. We describe how these tests were used during the first phase of the SARS outbreak in Toronto and offer some recommendations that may be useful if SARS returns. METHODS: We examined the results of all diagnostic laboratory tests used in 117 patients admitted to hospitals in Toronto who met the Health Canada criteria for suspect or probable SARS. Focusing on tests for SARS-CoV, we attempted to determine the optimal specimen types and timing of specimen collection. RESULTS: Diagnostic test results for SARS-CoV were available for 110 of the 117 patients. SARS-CoV was detected by means of reverse-transcriptase polymerase chain reaction (RT-PCR) in at least one specimen in 59 (54.1%) of 109 patients. Serologic test results of convalescent samples were positive in 50 (96.2%) of 52 patients for whom paired serum samples were collected during the acute and convalescent phases of the illness. Of the 110 patients, 78 (70.9%) had specimens that tested positive by means of RT-PCR, serologic testing or both methods. The proportion of RT-PCR test results that were positive was similar between patients who met the criteria for suspect SARS (50.8%, 95% confidence interval [CI] 38.4%-63.2%) and those who met the criteria for probable SARS (58.0%, 95% CI 44.2%-70.7%). SARS-CoV was detected in nasopharyngeal swabs in 33 (32.4%) of 102 patients, in stool specimens in 19 (63.3%) of 30 patients, and in specimens from the lower respiratory tract in 10 (58.8%) of 17 patients. INTERPRETATION: These findings suggest that the rapid diagnostic tests in use at the time of the initial outbreak lack sufficient sensitivity to be used clinically to rule out SARS. As tests for SARS-CoV continue to be optimized, evaluation of the clinical presentation and elucidation of a contact history must remain the cornerstone of SARS diagnosis. In patients with SARS, specimens taken from the lower respiratory tract and stool samples test positive by means of RT-PCR more often than do samples taken from other areas.

Adolescent↗

PubMed2004

Immunization with modified vaccinia virus Ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets.

Severe acute respiratory syndrome (SARS) caused by a newly identified coronavirus (SARS-CoV) is a serious emerging human infectious disease. In this report, we immunized ferrets (Mustela putorius furo) with recombinant modified vaccinia virus Ankara (rMVA) expressing the SARS-CoV spike (S) protein. Immunized ferrets developed a more rapid and vigorous neutralizing antibody response than control animals after challenge with SARS-CoV; however, they also exhibited strong inflammatory responses in liver tissue. Inflammation in control animals exposed to SARS-CoV was relatively mild. Thus, our data suggest that vaccination with rMVA expressing SARS-CoV S protein is associated with enhanced hepatitis.

Animals↗

PubMed2003

The Genome sequence of the SARS-associated coronavirus.

We sequenced the 29,751-base genome of the severe acute respiratory syndrome (SARS)-associated coronavirus known as the Tor2 isolate. The genome sequence reveals that this coronavirus is only moderately related to other known coronaviruses, including two human coronaviruses, HCoV-OC43 and HCoV-229E. Phylogenetic analysis of the predicted viral proteins indicates that the virus does not closely resemble any of the three previously known groups of coronaviruses. The genome sequence will aid in the diagnosis of SARS virus infection in humans and potential animal hosts (using polymerase chain reaction and immunological tests), in the development of antivirals (including neutralizing antibodies), and in the identification of putative epitopes for vaccine development.

3' Untranslated Regions↗

PubMed2003

Identification of new human coding steroid receptor RNA activator isoforms.

SRA is a steroid receptor co-activator which acts as a functional RNA and is classified as belonging to the growing family of functional non-coding RNAs. None of the different SRA transcripts described to date encode a detectable SRA protein following in vitro and in vivo translation experiments. We have identified three new SRA-RNA isoforms differing mainly from the originally cloned SRA by an extended 5(') extremity. These long SRA isoforms, able to encode a stable protein in vitro, led to the production in vivo of a nuclear protein when transfected into the MCF-7 human breast cancer cell line. Reverse-transcription polymerase chain reaction and Western blot analysis of RNA and protein extracts from different breast cancer cell lines confirmed the presence of endogenous coding SRA isoforms and their corresponding proteins. Our results demonstrate that full-length SRA-RNAs likely to encode stable proteins are widely expressed in breast cancer cell lines.

5' Flanking Region↗

PubMed2002

Emerging and re-emerging infectious diseases.

In human history, numerous infectious diseases have emerged and re-emerged. Aside from many others, the so-called 'exotic' agents in particular are a threat to our public health systems due to limited experience in case management and lack of appropriate resources. Many of these agents are zoonotic in origin and transmitted from animals to man either directly or via vectors. The reservoirs are often infected subclinically or asymptomatically and the distribution of the diseases basically reflects the range and the population dynamics of their reservoir hosts. As examples, emergence/re-emergence is discussed here for diseases caused by filoviruses, hantaviruses, paramyxoviruses, flaviviruses and Yersinia pestis. In addition, bioterrorism is addressed as one factor which has now to be considered in infectious disease emergence/re-emergence. Preparedness for known and unknown infectious diseases will be a top priority for our public health systems in the beginning of the millennium.

Animals↗