An expanded RT-PCR melting temperature coding assay to rapidly identify all known SARS-CoV-2 variants and sub-variants of concern.

The continued emergence of vaccine-resistant SARS-CoV-2 variants of concern (VOC) requires specific identification of each VOC as it arises. Here, we report an expanded version of our previously described sloppy molecular beacon (SMB) melting temperature (Tm) signature-based assay for VOCs, now modified to include detection of Delta (B.1.617.2) and Omicron (B.1.1.529) sub-variants. The SMB-VOC assay targets the signature codons 501, 484 and 452 in the SARS-CoV-2 spike protein which we show can specifically detect and differentiate all known VOCs including the Omicron subvariants (BA.1, BA.2, BA.2.12.1, BA.4/BA.5). The limit of detection (LOD) of the assay was 20, 22 and 36 genomic equivalents (GE) per reaction with the Delta, Omicron BA.1 and BA.2 respectively. Clinical validation of the 3-codon assay in the LC480 instrument showed the assay detected 94% (81/86) of the specimens as WT or VOCs and 6% (5/86) of the tests producing indeterminate results compared to sequencing. Sanger sequencing also failed for four samples. None of the specimens were incorrectly identified as WT or as a different VOC by our assay. Thus, excluding specimens with indeterminant results, the assay was 100% sensitive and 100% specific compared to Sanger sequencing for variant identification. This new assay concept can be easily expanded to add newer variants and can serve as a robust diagnostic tool for selecting appropriate monoclonal antibody therapy and rapid VOC surveillance.

Persistence of SARS-CoV-2 in saliva: Implications for late-stage diagnosis and infectious duration.

Saliva has been a COVID-19 diagnostic specimen of interest due to its simple collection, scalability, and yield. Yet COVID-19 testing and estimates of the infectious period remain largely based on nasopharyngeal and nasal swabs. We sought to evaluate whether saliva testing captured prolonged presence of SARS-CoV-2 and potential infectiousness later in the disease course. We conducted an observational study of symptomatic COVID-19 patients at University Hospital in Newark, NJ. Paired saliva and nasal specimens from 96 patients were analyzed, including longitudinal analysis of paired observations from 28 of these patients who had multiple time-points. Saliva detected significantly more cases of COVID-19 beyond 5 days (86.1% [99/115] saliva vs 48.7% [56/115] nasal, p-value < 0.001), 9 days (79.4% [50/63] saliva vs 36.5% [23/63] nasal, p-value < 0.001) and 14 days (71.4% [20/28] saliva vs 32.1% [9/28] nasal, p-value = 0.010) of symptoms. Additionally, saliva yielded lower cycle thresholds across all time periods, indicative of higher viral loads in saliva. In the longitudinal analysis, a log-rank analysis indicated that the survival curve for saliva was significantly different from the curve for nasal swabs (p<0.001) with a median survival time for saliva of 18 days compared to 13 days for nasal swabs. We additionally performed saliva viral cultures among a similar COVID-19 patient cohort and noted patients with positive saliva viral cultures between 7 to 28 days of symptoms. Findings from this study suggest that SARS-CoV-2 RNA persists longer and in higher abundance in saliva compared to nasal swabs, with potential of prolonged propagating virus. Testing saliva may thus increase yield for detecting potentially infectious virus even beyond the first five days of symptomatic COVID-19.

A Simple Reverse Transcriptase PCR Melting-Temperature Assay To Rapidly Screen for Widely Circulating SARS-CoV-2 Variants.

The increased transmission of SARS-CoV-2 variants of concern (VOC), which originated in the United Kingdom (B.1.1.7/alpha), South Africa (B1.351/beta), Brazil (P.1/gamma), the United States (B.1.427/429 or epsilon), and India (B.1.617.2/delta), requires a vigorous public health response, including real-time strain surveillance on a global scale. Although genome sequencing is the gold standard for identifying these VOCs, it is time-consuming and expensive. Here, we describe a simple, rapid, and high-throughput reverse transcriptase PCR (RT-PCR) melting-temperature (Tm) screening assay that identifies the first three major VOCs. RT-PCR primers and four sloppy molecular beacon (SMB) probes were designed to amplify and detect the SARS-CoV-2 N501Y (A23063T) and E484K (G23012A) mutations and their corresponding wild-type sequences. After RT-PCR, the VOCs were identified by a characteristic Tm of each SMB. Assay optimization and testing was performed with RNA from SARS-CoV-2 USA WA1/2020 (wild type [WT]), B.1.1.7, and B.1.351 variant strains. The assay was then validated using clinical samples. The limit of detection for both the WT and variants was 4 and 10 genomic copies/reaction for the 501- and 484-codon assays, respectively. The assay was 100% sensitive and 100% specific for identifying the N501Y and E484K mutations in cultured virus and in clinical samples, as confirmed by Sanger sequencing. We have developed an RT-PCR melt screening test for the major VOCs that can be used to rapidly screen large numbers of patient samples, providing an early warning for the emergence of these variants and a simple way to track their spread.

Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays.

BACKGROUND: Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. METHODS: We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. RESULTS: SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. CONCLUSION: eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

A Simple RT-PCR Melting temperature Assay to Rapidly Screen for Widely Circulating SARS-CoV-2 Variants.

BACKGROUND: The increased transmission of SARS-CoV-2 variants of concern (VOC) which originated in the United Kingdom (B.1.1.7), South Africa (B1.351), Brazil (P.1) and in United States (B.1.427/429) requires a vigorous public health response, including real time strain surveillance on a global scale. Although genome sequencing is the gold standard for identifying these VOCs, it is time consuming and expensive. Here, we describe a simple, rapid and high-throughput reverse-transcriptase PCR (RT-PCR) melting temperature (Tm) screening assay that identifies these three major VOCs. METHODS: RT-PCR primers and four sloppy molecular beacon (SMB) probes were designed to amplify and detect the SARS-CoV-2 N501Y (A23063T) and E484K (G23012A) mutations and their corresponding wild type sequences. After RT-PCR, the VOCs were identified by a characteristic Tm of each SMB. Assay optimization and testing was performed with RNA from SARS-CoV-2 USA WA1/2020 (WT), a B.1.17 and a B.1.351 variant strains. The assay was then validated using clinical samples. RESULTS: The limit of detection (LOD) for both the WT and variants was 4 and 10 genomic copies/reaction for the 501 and 484 codon assays, respectively. The assay was 100% sensitive and 100% specific for identifying the N501Y and E484K mutations in cultured virus and in clinical samples as confirmed by Sanger sequencing. CONCLUSION: We have developed an RT-PCR melt screening test for the three major VOCs which can be used to rapidly screen large numbers of patient samples providing an early warning for the emergence of these variants and a simple way to track their spread.

Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays.

BACKGROUND: Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. METHODS: We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream RT-PCR testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. RESULTS: SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log 10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on vero-E6 cell lines was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. CONCLUSION: eNAT and similar guanidinium thiocyanate-based media may be of value for transport, preservation, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 Test.

Nucleic acid amplification tests (NAATs) are the primary means of identifying acute infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Accurate and fast test results may permit more efficient use of protective and isolation resources and allow rapid therapeutic interventions. We evaluated the analytical and clinical performance characteristics of the Xpert Xpress SARS-CoV-2 (Xpert) test, a rapid, automated molecular test for SARS-CoV-2. Analytical sensitivity and specificity/interference were assessed with infectious SARS-CoV-2; other infectious coronavirus species, including SARS-CoV; and 85 nasopharyngeal swab specimens positive for other respiratory viruses, including endemic human coronaviruses (hCoVs). Clinical performance was assessed using 483 remnant upper- and lower-respiratory-tract specimens previously analyzed by standard-of-care (SOC) NAATs. The limit of detection of the Xpert test was 0.01 PFU/ml. Other hCoVs, including Middle East respiratory syndrome coronavirus, were not detected by the Xpert test. SARS-CoV, a closely related species in the subgenus Sarbecovirus, was detected by a broad-range target (E) but was distinguished from SARS-CoV-2 (SARS-CoV-2-specific N2 target). Compared to SOC NAATs, the positive agreement of the Xpert test was 219/220 (99.5%), and the negative agreement was 250/261 (95.8%). A third tie-breaker NAAT resolved all but three of the discordant results in favor the Xpert test. The Xpert test provided sensitive and accurate detection of SARS-CoV-2 in a variety of upper- and lower-respiratory-tract specimens. The high sensitivity and short time to results of approximately 45 min may impact patient management.

Multiplex Detection of Three Select Agents Directly from Blood by Use of the GeneXpert System.

Francisella tularensis, Bacillus anthracis, and Yersinia pestis are tier 1 select agents with the potential to rapidly cause severe disease. Rapid detection of these bacteria from patient samples at the point of care could contribute to improved clinical outcomes in the event of a bioterrorism attack. A multiplex nested PCR assay for detection of F. tularensis, B. anthracis, and Y. pestis directly from patient blood samples was developed using the GeneXpert system. The multiplex GeneXpert cartridge-based assay includes all necessary sample processing and amplification reagents. Blood samples spiked with different numbers of CFU were used to measure the analytical limit of detection (LOD) and dynamic range. Sensitivity was determined by testing spiked blood samples and negative-control blood in a blind manner. Specificity was determined by testing against nontarget pathogens and blood samples from clinical patients. The assay LOD was 8.5 CFU/ml for F. tularensis, 10 CFU/ml for B. anthracis, and 4.5 CFU/ml for Y. pestis The sensitivity was 100% at the LOD for all three select agent bacteria in spiked patient blood samples. The assay specificity was 100% when it was tested against both nontarget pathogens and clinical patient blood samples. The total assay time was approximately 100 min. This automated assay, which is suitable for use at the point of care, identifies three select agents directly in blood without the need for enrichment with a high sensitivity within 100 min. This assay may enable rapid detection and treatment of patients infected with the target organisms in the event of a bioterrorism attack.

An Improved Tobacco Mosaic Virus (TMV)-Conjugated Multiantigen Subunit Vaccine Against Respiratory Tularemia.

Francisella tularensis, the causative agent of the fatal human disease known as tularemia is classified as a Category A Select Agent by the Centers for Disease Control. No licensed vaccine is currently available for prevention of tularemia in the United States. Previously, we published that a tri-antigen tobacco mosaic virus (TMV) vaccine confers 50% protection in immunized mice against respiratory tularemia caused by F. tularensis. In this study, we refined the TMV-vaccine formulation to improve the level of protection in immunized C57BL/6 mice against respiratory tularemia. We developed a tetra-antigen vaccine by conjugating OmpA, DnaK, Tul4, and SucB proteins of Francisella to TMV. CpG was also included in the vaccine formulation as an adjuvant. Primary intranasal (i.n.) immunization followed by two booster immunizations with the tetra-antigen TMV vaccine protected 100% mice against i.n. 10LD100 challenges dose of F. tularensis live vaccine strain (LVS). Mice receiving three immunization doses of tetra-antigen TMV vaccine showed only transient body weight loss, cleared the infection rapidly, and showed minimal histopathological lesions in lungs, liver, and spleen following a lethal respiratory challenge with F. tularensis LVS. Mice immunized with the tetra-antigen TMV vaccine also induced strong ex vivo recall responses and were protected against a lethal challenge as late as 163 days post-primary immunization. Three immunization with the tetra-antigen TMV vaccine also induced a stronger humoral immune response predominated by IgG1, IgG2b, and IgG2c antibodies than mice receiving only a single or two immunizations. Remarkably, a single dose protected 40% of mice, while two doses protected 80% of mice from lethal pathogen challenge. Immunization of Interferon-gamma (IFN-γ)-deficient mice with the tetra-antigen TMV vaccine demonstrated an absolute requirement of IFN-γ for the generation of protective immune response against a lethal respiratory challenge with F. tularensis LVS. Collectively, this study further demonstrates the feasibility of TMV as an efficient platform for the delivery of multiple F. tularensis antigens and that tetra-antigen TMV vaccine formulation provides complete protection, and induces long-lasting protective and memory immune responses against respiratory tularemia caused by F. tularensis LVS.

Development of a Multivalent Subunit Vaccine against Tularemia Using Tobacco Mosaic Virus (TMV) Based Delivery System.

Francisella tularensis is a facultative intracellular pathogen, and is the causative agent of a fatal human disease known as tularemia. F. tularensis is classified as a Category A Biothreat agent by the CDC based on its use in bioweapon programs by several countries in the past and its potential to be used as an agent of bioterrorism. No licensed vaccine is currently available for prevention of tularemia. In this study, we used a novel approach for development of a multivalent subunit vaccine against tularemia by using an efficient tobacco mosaic virus (TMV) based delivery platform. The multivalent subunit vaccine was formulated to contain a combination of F. tularensis protective antigens: OmpA-like protein (OmpA), chaperone protein DnaK and lipoprotein Tul4 from the highly virulent F. tularensis SchuS4 strain. Two different vaccine formulations and immunization schedules were used. The immunized mice were challenged with lethal (10xLD100) doses of F. tularensis LVS on day 28 of the primary immunization and observed daily for morbidity and mortality. Results from this study demonstrate that TMV can be used as a carrier for effective delivery of multiple F. tularensis antigens. TMV-conjugate vaccine formulations are safe and multiple doses can be administered without causing any adverse reactions in immunized mice. Immunization with TMV-conjugated F. tularensis proteins induced a strong humoral immune response and protected mice against respiratory challenges with very high doses of F. tularensis LVS. This study provides a proof-of-concept that TMV can serve as a suitable platform for simultaneous delivery of multiple protective antigens of F. tularensis. Refinement of vaccine formulations coupled with TMV-targeting strategies developed in this study will provide a platform for development of an effective tularemia subunit vaccine as well as a vaccination approach that may broadly be applicable to many other bacterial pathogens.

Preclinical testing of a vaccine candidate against tularemia.

Tularemia is caused by a gram-negative, intracellular bacterial pathogen, Francisella tularensis (Ft). The history weaponization of Ft in the past has elevated concerns that it could be used as a bioweapon or an agent of bioterrorism. Since the discovery of Ft, three broad approaches adopted for tularemia vaccine development have included inactivated, live attenuated, or subunit vaccines. Shortcomings in each of these approaches have hampered the development of a suitable vaccine for prevention of tularemia. Recently, we reported an oxidant sensitive mutant of Ft LVS in putative EmrA1 (FTL_0687) secretion protein. The emrA1 mutant is highly sensitive to oxidants, attenuated for intramacrophage growth and virulence in mice. We reported that EmrA1 contributes to oxidant resistance by affecting the secretion of antioxidant enzymes SodB and KatG. This study investigated the vaccine potential of the emrA1 mutant in prevention of respiratory tularemia caused by Ft LVS and the virulent SchuS4 strain in C57BL/6 mice. We report that emrA1 mutant is safe and can be used at an intranasal (i. n.) immunization dose as high as 1x106 CFU without causing any adverse effects in immunized mice. The emrA1 mutant is cleared by vaccinated mice by day 14-21 post-immunization, induces minimal histopathological lesions in lungs, liver and spleen and a strong humoral immune response. The emrA1 mutant vaccinated mice are protected against 1000-10,000LD100 doses of i.n. Ft LVS challenge. Such a high degree of protection has not been reported earlier against respiratory challenge with Ft LVS using a single immunization dose with an attenuated mutant generated on Ft LVS background. The emrA1 mutant also provides partial protection against i.n. challenge with virulent Ft SchuS4 strain in vaccinated C57BL/6 mice. Collectively, our results further support the notion that antioxidants of Ft may serve as potential targets for development of effective vaccines for prevention of tularemia.

EmrA1 membrane fusion protein of Francisella tularensis LVS is required for resistance to oxidative stress, intramacrophage survival and virulence in mice.

Francisella tularensis is a category A biodefence agent that causes a fatal human disease known as tularaemia. The pathogenicity of F. tularensis depends on its ability to persist inside host immune cells primarily by resisting an attack from host-generated reactive oxygen and nitrogen species (ROS/RNS). Based on the ability of F. tularensis to resist high ROS/RNS levels, we have hypothesized that additional unknown factors act in conjunction with known antioxidant defences to render ROS resistance. By screening a transposon insertion library of F. tularensis LVS in the presence of hydrogen peroxide, we have identified an oxidant-sensitive mutant in putative EmrA1 (FTL_0687) secretion protein. The results demonstrate that the emrA1 mutant is highly sensitive to oxidants and several antimicrobial agents, and exhibits diminished intramacrophage growth that can be restored to wild-type F. tularensis LVS levels by either transcomplementation, inhibition of ROS generation or infection in NADPH oxidase deficient (gp91Phox(-/-)) macrophages. The emrA1 mutant is attenuated for virulence, which is restored by infection in gp91Phox(-/-) mice. Further, EmrA1 contributes to oxidative stress resistance by affecting secretion of Francisella antioxidant enzymes SodB and KatG. This study exposes unique links between transporter activity and the antioxidant defence mechanisms of F. tularensis.

Repression of inflammasome by Francisella tularensis during early stages of infection.

Francisella tularensis is an important human pathogen responsible for causing tularemia. F. tularensis has long been developed as a biological weapon and is now classified as a category A agent by the Centers for Disease Control because of its possible use as a bioterror agent. F. tularensis represses inflammasome; a cytosolic multi-protein complex that activates caspase-1 to produce proinflammatory cytokines IL-1ß and IL-18. However, the Francisella factors and the mechanisms through which F. tularensis mediates these suppressive effects remain relatively unknown. Utilizing a mutant of F. tularensis in FTL_0325 gene, this study investigated the mechanisms of inflammasome repression by F. tularensis. We demonstrate that muted IL-1ß and IL-18 responses generated in macrophages infected with F. tularensis live vaccine strain (LVS) or the virulent SchuS4 strain are due to a predominant suppressive effect on TLR2-dependent signal 1. Our results also demonstrate that FTL_0325 of F. tularensis impacts proIL-1ß expression as early as 2 h post-infection and delays activation of AIM2 and NLRP3-inflammasomes in a TLR2-dependent fashion. An enhanced activation of caspase-1 and IL-1ß observed in FTL_0325 mutant-infected macrophages at 24 h post-infection was independent of both AIM2 and NLRP3. Furthermore, F. tularensis LVS delayed pyroptotic cell death of the infected macrophages in an FTL_0325-dependent manner during the early stages of infection. In vivo studies in mice revealed that suppression of IL-1ß by FTL_0325 early during infection facilitates the establishment of a fulminate infection by F. tularensis. Collectively, this study provides evidence that F. tularensis LVS represses inflammasome activation and that F. tularensis-encoded FTL_0325 mediates this effect.

Identification of a live attenuated vaccine candidate for tularemia prophylaxis.

Francisella tularensis is the causative agent of a fatal human disease, tularemia. F. tularensis was used in bioweapon programs in the past and is now classified as a category A select agent owing to its possible use in bioterror attacks. Despite over a century since its discovery, an effective vaccine is yet to be developed. In this study four transposon insertion mutants of F. tularensis live vaccine strain (LVS) in Na/H antiporter (FTL_0304), aromatic amino acid transporter (FTL_0291), outer membrane protein A (OmpA)-like family protein (FTL_0325) and a conserved hypothetical membrane protein gene (FTL_0057) were evaluated for their attenuation and protective efficacy against F. tularensis SchuS4 strain. All four mutants were 100-1000 fold attenuated for virulence in mice than parental F. tularensis. Except for the FTL_0304, single intranasal immunization with the other three mutants provided 100% protection in BALB/c mice against intranasal challenge with virulent F. tularensis SchuS4. Differences in the protective ability of the FTL_0325 and FTL_0304 mutant which failed to provide protection against SchuS4 were investigated further. The results indicated that an early pro-inflammatory response and persistence in host tissues established a protective immunity against F. tularensis SchuS4 in the FTL_0325 immunized mice. No differences were observed in the levels of serum IgG antibodies amongst the two vaccinated groups. Recall response studies demonstrated that splenocytes from the FTL_0325 mutant immunized mice induced significantly higher levels of IFN-γ and IL-17 cytokines than the FTL_0304 immunized counterparts indicating development of an effective memory response. Collectively, this study demonstrates that persistence of the vaccine strain together with its ability to induce an early pro-inflammatory innate immune response and strong memory responses can discriminate between successful and failed vaccinations against tularemia. This study describes a live attenuated vaccine which may prove to be an ideal vaccine candidate for prevention of respiratory tularemia.

BB0844, an RpoS-regulated protein, is dispensable for Borrelia burgdorferi infectivity and maintenance in the mouse-tick infectious cycle.

The genome of Borrelia burgdorferi, the causative agent of Lyme disease, is comprised of a large linear chromosome and numerous smaller linear and circular plasmids. B. burgdorferi exhibits substantial genomic variation, and previous studies revealed genotype-specific variation at the right chromosomal telomere. A correlation has also been established between genotype and invasiveness. The correlation between chromosome length and genotype and between genotype and invasiveness suggested that a gene(s) at the right chromosome telomere may be required for virulence. Of particular interest was bb0844, an RpoS-regulated gene at the right telomere, the expression of which is induced when the spirochete undergoes adaptation to the mammalian host. The structure of the right chromosomal telomere was examined in 53 B. burgdorferi clinical isolates of various genotypes. Four distinct patterns were observed for bb0844: (i) chromosomal localization, (ii) plasmid localization, (iii) presence on both chromosome and plasmid, and (iv) complete absence. These patterns correlated with the B. burgdorferi genotype. On the basis of available sequence data, we propose a mechanism for the genomic rearrangements that accounts for the variability in bb0844 genomic localization. To further explore the role of BB0844 in the spirochete life cycle, a bb0844 deletion mutant was constructed by allelic exchange, and the viability of wild-type and bb0844 deletion mutants was examined in an experimental mouse-tick infection model. The bb0844 mutant was fully infectious in C3H/HeJ mice by either needle inoculation or tick transmission with B. burgdorferi-infected Ixodes scapularis larvae. Naïve larval ticks acquired both wild-type and mutant spirochetes with equal efficiency from B. burgdorferi-infected mice. The results demonstrate that BB0844 is not required for spirochete viability, pathogenicity, or maintenance in the tick vector or the mammalian host. At present, a defined role for BB0844 in B. burgdorferi cannot be ascertained.

Role of Mga in group A streptococcal infection at the skin epithelium.

Group A streptococci (GAS) primarily cause infection at epithelial tissue sites of its human host. The role of the transcriptional regulator Mga in a humanized mouse model for superficial skin infection was investigated. Inactivation of mga in a skin strain (Alab49) led to loss of virulence. The Deltamga mutant displayed >100-fold decrease in emm (pam) transcript levels, and loss of bacterial-bound plasmin activity. A slight decrease in speB transcription, accompanied by a partial decrease in cysteine protease activity but no change in PrtF2 degradation, was also observed. Mga had no effect on transcription of nra, Nra-regulated pilus genes (cpa, fctA) or other FCT-region genes (msmR, prtF2). Combined with findings on other Alab49 mutants, data show that several essential virulence genes are regulated by Mga or Nra, but not both, implying that any coordinated response during skin infection likely operates at a higher level of transcriptional control. Mga was required for bacterial autoaggregation and biofilm-like growth on an abiotic surface; however, aggregation and biofilm formation have only partial overlap with the skin virulence phenotype. Findings on numerous phenotypes for 7 mutants constructed on the same genetic background yield a detailed, integrated model for GAS pathogenesis at the skin.

Toxin production by Aeromonas sobria in natural environments: river water vs. seawater.

Aeromonas are water-borne pathogens. They are halotolerant, which means that they can survive in environments whose salt content corresponds to that of seawater (3.0% NaCl). However, the presence of Aeromonas in seawater is extremely rare compared with that in river water. In this study, we tested the ability of Aeromonas sobria to produce toxins in river water and seawater. First, we cultured A. sobria on skim milk agar plates supplemented with either river water (SARW) or seawater (SASW). The bacteria grew on both plates. A clear zone around the bacteria was generated in SARW. However, such a zone was not observed in SASW, suggesting that proteases were not generated in SASW. Subsequently, we cultured A. sobria in a nutrient broth supplemented with either river water (NRW) or with seawater (NSW), and examined the protease activity of their culture supernatants. The protease activity of the culture supernatant from NSW was extremely low compared to that from NRW. The immunoblotting analysis showed that serine protease (ASP) was not produced by the culture in NSW. By contrast, aerolysin-like hemolysin was produced in all conditions examined in this study. This indicates that the salinity of water is deeply involved in the production of ASP by A. sobria.

Sequence analysis and evolution of group B rotaviruses.

Human group B rotaviruses were isolated from hospitalized patients in Bangladesh between July 2003 and December 2004. Phylogenetic analyses of the gene segments encoding the hemagglutinin (VP4), glycoprotein (VP7) and RNA-binding protein (NSP2) of group B rotaviruses showed that Bangladeshi strains were more similar to the Indian strains than to the prototype Chinese strains. Moreover, all human strains were clustered together and were distantly related to the animal strains. With limited sequence data, the evolutionary rate of the glycoproteins (VP7) of human group B rotaviruses was estimated to be 1.57x10(-3) nucleotide substitutions/(siteyear), which was comparable to other rapidly evolving RNA viruses. The most recent common ancestor (MRCA) of the extant human group B rotaviruses was calculated to date to around 1976.

Role of divalent metal cations in ATP hydrolysis catalyzed by the hepatitis C virus NS3 helicase: magnesium provides a bridge for ATP to fuel unwinding.

This study investigates the role of magnesium ions in coupling ATP hydrolysis to the nucleic acid unwinding catalyzed by the NS3 protein encoded by the hepatitis C virus (HCV). Analyses of steady-state ATP hydrolysis rates at various RNA and magnesium concentrations were used to determine values for the 15 dissociation constants describing the formation of a productive enzyme-metal-ATP-RNA complex and the four rate constants describing hydrolysis of ATP by the possible enzyme-ATP complexes. These values coupled with direct binding studies, specificity studies and analyses of site-directed mutants reveal only one ATP binding site on HCV helicase centered on the catalytic base Glu291. An adjacent residue, Asp290, binds a magnesium ion that forms a bridge to ATP, reorienting the nucleotide in the active site. RNA stimulates hydrolysis while decreasing the affinity of the enzyme for ATP, magnesium, and MgATP. The binding scheme described here explains the unusual regulation of the enzyme by ATP that has been reported previously. Binding of either free magnesium or free ATP to HCV helicase competes with MgATP, the true fuel for helicase movements, and leads to slower hydrolysis and nucleic acid unwinding.