Evaluation of Prenatal Hepatitis C Virus Prevalence Using Universal Screening, and Linkage to Care in a Real-World Setting in Ontario.

OBJECTIVES: International infectious disease/obstetrical societies have recently recommended universal hepatitis C virus (HCV) prenatal screening and these same recommendations are forthcoming in Canada. At present, there is no formal analysis of universal HCV screening or linkage to care of pregnant people in Ontario. The objectives of our study were to determine the seroprevalence of HCV using 2 different methods to evaluate universal screening, as well as identify opportunities that may improve linkage to care. METHODS: To assess seroprevalence in a large urban area, we aimed to test 12 000 de-identified samples submitted for prenatal HIV testing in the catchment area of Toronto Public Health for HCV antibodies. Then, to assess the seroprevalence as well as the operational impact and follow-up in a real-world setting, we completed a Quality Improvement Project (QIP) for 1 year at a large tertiary care obstetrical centre in London, Ontario. RESULTS: From 2019 to 2021, 11 999 de-identified samples were screened from Toronto with a seroprevalence of 0.40 (95% CI 0.29-0.53). In London, 5771 people were screened in 2021 with a seroprevalence of 0.55% (95% CI 0.38-0.78). Taken together, those aged 26-35 years had the highest positivity; in the QIP, 9% had no documented risk factor, and 59% of individuals were not linked to the next step in HCV care. CONCLUSIONS: HCV prenatal seroprevalence in Ontario is comparable to hepatitis B virus, and ∼15-30-fold higher than HIV. Diagnosis in pregnancy is critical to facilitate referrals for treatment between pregnancies and could increase screening among children born to positive women.

Evaluation of Real and Perceived Risk to Health Care Workers Caring for Patients With the Omicron Variant of the SARS-CoV-2 Virus in Surgery and Obstetrics.

OBJECTIVES: The Omicron variant of the SARS-CoV-2 virus is described as more contagious than previous variants. We sought to assess risk to health care workers (HCWs) caring for patients with COVID-19 in surgical/obstetrical settings, and the perception of risk among this group. METHODS: From January to April 2022, reverse transcription polymerase chain reaction was used to detect the presence of SARS-CoV-2 viral ribonucleic acid in patient, environmental (floor, equipment, passive air) samples, and HCWs' masks (inside surface) during urgent surgery or obstetrical delivery for patients with SARS-CoV-2 infection. The primary outcome was the proportion of HCWs' masks testing positive. Results were compared with our previous cross-sectional study involving obstetrical/surgical patients with earlier variants (2020-2021). HCWs completed a risk perception electronic questionnaire. RESULTS: Eleven patients were included: 3 vaginal births and 8 surgeries. In total, 5/108 samples (5%) tested positive (SARS-CoV-2 Omicron) viral ribonucleic acid: 2/5 endotracheal tubes, 1/22 floor samples, 1/4 patient masks, and 1 nasal probe. No samples from the HCWs' masks (0/35), surgical equipment (0/10), and air (0/11) tested positive. No significant differences were found between the Omicron and 2020/21 patient groups' positivity rates (Mann-Whitney U test, P = 0.838) or the level of viral load from the nasopharyngeal swabs (P = 0.405). Nurses had a higher risk perception than physicians (P = 0.038). CONCLUSION: No significant difference in contamination rates was found between SARS-CoV-2 Omicron BA.1 and previous variants in surgical/obstetrical settings. This is reassuring as no HCW mask was positive and no HCW tested positive for COVID-19 post-exposure.

Cystic neutrophilic granulomatous mastitis: sensitivity and specificity of 16s rRNA and Sanger sequencing for Corynebacterium spp.

AIMS: Cystic neutrophilic granulomatous mastitis (CNGM) is a subtype of granulomatous mastitis (GM) associated with Corynebacterium spp infection. We aimed to analyse the prevalence of Corynebacteria in CNGM and non-CNGM cases. METHODS: Breast specimens diagnosed as granulomatous inflammation between 2010 and 2020 were reviewed to identify a CNGM cohort and a non-CNGM cohort. Polymerase chain reaction-based identification of Corynebacteria by 16S ribosomal RNA (16S rRNA) primers, followed by confirmatory Sanger sequencing (SS), was performed on all cases. Clinical, radiological and microbiology data were retrieved from the electronic patient records. RESULTS: Twenty-eight CNGM cases and 19 non-CNGM cases were identified. Compared with the non-CNGM cohort, patients in the CNGM cohort were more likely to be multiparous (p=0.01), breast feeding (p=0.01) and presenting with a larger breast mass (p<0.01), spontaneous drainage (p=0.05) and skin irritation (p<0.01). No significant difference in the prevalence of Corynebacteria between the cohorts (7% vs 11%, p=0.68) by microbiological culture was identified. Compared with microbiology culture, the sensitivity and specificity of each Corynebacterial detection method were 50% and 81% for Gram stain, and 25% and 100% for 16S rRNA combined with SS. Regardless of the diagnosis, patients positive for Corynebacteria were more likely to have a persistent disease (p<0.01). CONCLUSION: CNGM presents as a large symptomatic breast mass in multiparous breastfeeding women. The importance of adequate sampling and repeated microbiology culture in conjunction with sequencing on all GM cases with persistent disease is paramount.

BCG administration promotes the long-term protection afforded by a single-dose intranasal adenovirus-based SARS-CoV-2 vaccine.

Recent publications have explored intranasal (i.n.) adenovirus-based (Ad) vaccines as an effective strategy for SARS-CoV-2 in pre-clinical models. However, the effects of prior immunizations and infections have yet to be considered. Here, we investigate the immunomodulatory effects of Mycobacterium bovis BCG pre-immunization followed by vaccination with an S-protein-expressing i.n. Ad, termed Ad(Spike). While i.n. Ad(Spike) retains some protective effect after 6 months, a single administration of BCG-Danish prior to Ad(Spike) potentiates its ability to control viral replication of the B.1.351 SARS-CoV-2 variant within the respiratory tract. Though BCG-Danish did not affect Ad(Spike)-generated humoral immunity, it promoted the generation of cytotoxic/Th1 responses over suppressive FoxP3+ TREG cells in the lungs of infected mice. Thus, this vaccination strategy may prove useful in limiting future pandemics by potentiating the long-term efficacy of mucosal vaccines within the context of the widely distributed BCG vaccine.

Lachnoanaerobaculum orale bacteremia in a patient with acute myeloid leukemia and stomatitis: An emerging pathogen.

Background: Lachnoanaerobaculum orale is a newly described, obligately anaerobic gram-positive bacillus. The first report of invasive disease caused by L. orale was described in a patient with acute lymphocytic leukemia following systematic chemotherapy. Here we describe another case of L. orale bacteremia in a patient with a hematologic malignancy following chemotherapy-induced neutropenia. Methods: We present a case of a 46-year-old woman with a recent diagnosis of AML who presented to Sunnybrook Health Sciences Center with febrile neutropenia following induction chemotherapy with daunorubicin-cytarabine (3 +7 regimen) with Gemtuzumab and Ozogamycin. Despite being on intravenous pipercillin-tazobactam she remained febrile. Following our clinical assessment and investigations, potential sources of infection included a swollen digit and severe mucositis. Results: One blood culture from admission grew Lachnoanaerobaculum orale in the anaerobic bottle, identified by Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF). The isolate also underwent whole-genome sequencing using methods that have been described previously. Results demonstrated the organism was susceptible to cefoxitin, clindamycin, meropenem, metronidazole, penicillin, and piperacillin-tazobactam. We concluded that the source of this patient's bloodstream infection to be chemotherapy-induced stomatitis. Conclusion: With the increasing use of intensive immunosuppressive regimens and hematopoietic stem cell transplantation for patients with hematologic malignancies, there has been an increase in the incidence and detection of bloodstream infections due to anaerobic organisms. This is only the second case report of L. orale bacteremia, highlighting its emerging role as an opportunistic pathogen in immunocompromised patients.

Sphingosine Kinases Promote Ebola Virus Infection and Can Be Targeted to Inhibit Filoviruses, Coronaviruses, and Arenaviruses Using Late Endocytic Trafficking to Enter Cells.

Entry of enveloped viruses in host cells requires the fusion of viral and host cell membranes, a process that is facilitated by viral fusion proteins protruding from the viral envelope. These viral fusion proteins need to be triggered by host factors, and for some viruses, this event occurs inside endosomes and/or lysosomes. Consequently, these 'late-penetrating viruses' must be internalized and delivered to entry-conducive intracellular vesicles. Because endocytosis and vesicular trafficking are tightly regulated cellular processes, late-penetrating viruses also depend on specific host proteins for efficient delivery to the site of fusion, suggesting that these could be targeted for antiviral therapy. In this study, we investigated a role for sphingosine kinases (SKs) in viral entry and found that chemical inhibition of sphingosine kinase 1 (SK1) and/or SK2 and knockdown of SK1/2 inhibited entry of Ebola virus (EBOV) into host cells. Mechanistically, inhibition of SK1/2 prevented EBOV from reaching late-endosomes and lysosomes that contain the EBOV receptor, Niemann Pick C1 (NPC1). Furthermore, we present evidence that suggests that the trafficking defect caused by SK1/2 inhibition occurs independently of sphingosine-1-phosphate (S1P) signaling through cell-surface S1P receptors. Lastly, we found that chemical inhibition of SK1/2 prevents entry of other late-penetrating viruses, including arenaviruses and coronaviruses, and inhibits infection by replication-competent EBOV and SARS-CoV-2 in Huh7.5 cells. In sum, our results highlight an important role played by SK1/2 in endocytic trafficking, which can be targeted to inhibit entry of late-penetrating viruses and could serve as a starting point for the development of broad-spectrum antiviral therapeutics.

Altered microRNA Transcriptome in Cultured Human Airway Cells upon Infection with SARS-CoV-2.

Numerous proteomic and transcriptomic studies have been carried out to better understand the current multi-variant SARS-CoV-2 virus mechanisms of action and effects. However, they are mostly centered on mRNAs and proteins. The effect of the virus on human post-transcriptional regulatory agents such as microRNAs (miRNAs), which are involved in the regulation of 60% of human gene activity, remains poorly explored. Similar to research we have previously undertaken with other viruses such as Ebola and HIV, in this study we investigated the miRNA profile of lung epithelial cells following infection with SARS-CoV-2. At the 24 and 72 h post-infection time points, SARS-CoV-2 did not drastically alter the miRNome. About 90% of the miRNAs remained non-differentially expressed. The results revealed that miR-1246, miR-1290 and miR-4728-5p were the most upregulated over time. miR-196b-5p and miR-196a-5p were the most downregulated at 24 h, whereas at 72 h, miR-3924, miR-30e-5p and miR-145-3p showed the highest level of downregulation. In the top significantly enriched KEGG pathways of genes targeted by differentially expressed miRNAs we found, among others, MAPK, RAS, P13K-Akt and renin secretion signaling pathways. Using RT-qPCR, we also showed that SARS-CoV-2 may regulate several predicted host mRNA targets involved in the entry of the virus into host cells (ACE2, TMPRSS2, ADAM17, FURIN), renin-angiotensin system (RAS) (Renin, Angiotensinogen, ACE), innate immune response (IL-6, IFN1ß, CXCL10, SOCS4) and fundamental cellular processes (AKT, NOTCH, WNT). Finally, we demonstrated by dual-luciferase assay a direct interaction between miR-1246 and ACE-2 mRNA. This study highlights the modulatory role of miRNAs in the pathogenesis of SARS-CoV-2.

Detection of SARS-CoV-2 contamination in the operating room and birthing room setting: a cross-sectional study.

BACKGROUND: The exposure risks to front-line health care workers caring for patients with SARS-CoV-2 infection undergoing surgery or obstetric delivery are unclear, and an understanding of sample types that may harbour virus is important for evaluating risk. We sought to determine whether SARS-CoV-2 viral RNA from patients with SARS-CoV-2 infection undergoing surgery or obstetric delivery was present in the peritoneal cavity of male and female patients, in the female reproductive tract, in the environment of the surgery or delivery suite (surgical instruments or equipment used, air or floors), and inside the masks of the attending health care workers. METHODS: We conducted a cross-sectional study from November 2020 to May 2021 at 2 tertiary academic Toronto hospitals, during urgent surgeries or obstetric deliveries for patients with SARS-CoV-2 infection. The presence of SARS-CoV-2 viral RNA in patient, environmental and air samples was identified by real-time reverse transcription polymerase chain reaction (RT-PCR). Air samples were collected using both active and passive sampling techniques. The primary outcome was the proportion of health care workers' masks positive for SARS-CoV-2 RNA. We included adult patients with positive RT-PCR nasal swab undergoing obstetric delivery or urgent surgery (from across all surgical specialties). RESULTS: A total of 32 patients (age 20-88 yr) were included. Nine patients had obstetric deliveries (6 cesarean deliveries), and 23 patients (14 male) required urgent surgery from the orthopedic or trauma, general surgery, burn, plastic surgery, cardiac surgery, neurosurgery, vascular surgery, gastroenterology and gynecologic oncology divisions. SARS-CoV-2 RNA was detected in 20 of 332 (6%) patient and environmental samples collected: 4 of 24 (17%) patient samples, 5 of 60 (8%) floor samples, 1 of 54 (2%) air samples, 10 of 23 (43%) surgical instrument or equipment samples, 0 of 24 cautery filter samples and 0 of 143 (95% confidence interval 0-0.026) inner surface of mask samples. INTERPRETATION: During the study period of November 2020 to May 2021, we found evidence of SARS-CoV-2 RNA in a small but important number of samples obtained in the surgical and obstetric operative environment. The finding of no detectable virus inside the masks worn by the health care teams would suggest a low risk of infection for health care workers using appropriate personal protective equipment.

Immunogenicity of convalescent and vaccinated sera against clinical isolates of ancestral SARS-CoV-2, Beta, Delta, and Omicron variants.

Background: SARS-CoV-2 Omicron variant of concern (VOC) has evolved multiple mutations within the spike protein, raising concerns of increased antibody evasion. In this study, we assessed the neutralization potential of COVID-19 convalescent sera and sera from vaccinated individuals against ancestral SARS-CoV-2 and VOCs. Methods: The neutralizing activity of sera from 65 coronavirus disease (COVID-19) vaccine recipients and convalescent individuals against clinical isolates of ancestral SARS-CoV-2 and Beta, Delta, and Omicron VOCs was assessed using a micro-neutralization assay. Findings: Convalescent sera from unvaccinated individuals infected by the ancestral virus demonstrated reduced neutralization against Beta and Omicron VOCs. Sera from individuals that received three doses of the Pfizer or Moderna vaccines demonstrated reduced neutralization of the Omicron variant relative to ancestral SARS-CoV-2. Sera from individuals that were naturally infected with ancestral SARS-CoV-2 and subsequently received two doses of the Pfizer vaccine induced significantly higher neutralizing antibody levels against ancestral virus and all VOCs. Infection alone, either with ancestral SARS-CoV-2 or the Delta variant, was not sufficient to induce high neutralizing antibody titers against Omicron. Conclusions: In summary, we demonstrate that convalescent and vaccinated sera display varying levels of SARS-CoV-2 VOC neutralization. Data from this study will inform booster vaccination strategies against SARS-CoV-2 VOCs. Funding: This research was funded by the Canadian Institutes of Health Research (CIHR). VIDO receives operational funding from the Government of Saskatchewan through Innovation Saskatchewan and the Ministry of Agriculture and from the Canada Foundation for Innovation through the Major Science Initiatives for its CL3 facility.

Altered microRNA Transcriptome in Cultured Human Liver Cells upon Infection with Ebola Virus.

Ebola virus (EBOV) is a virulent pathogen, notorious for inducing life-threatening hemorrhagic fever, that has been responsible for several outbreaks in Africa and remains a public health threat. Yet, its pathogenesis is still not completely understood. Although there have been numerous studies on host transcriptional response to EBOV, with an emphasis on the clinical features, the impact of EBOV infection on post-transcriptional regulatory elements, such as microRNAs (miRNAs), remains largely unexplored. MiRNAs are involved in inflammation and immunity and are believed to be important modulators of the host response to viral infection. Here, we have used small RNA sequencing (sRNA-Seq), qPCR and functional analyses to obtain the first comparative miRNA transcriptome (miRNome) of a human liver cell line (Huh7) infected with one of the following three EBOV strains: Mayinga (responsible for the first Zaire outbreak in 1976), Makona (responsible for the West Africa outbreak in 2013-2016) and the epizootic Reston (presumably innocuous to humans). Our results highlight specific miRNA-based immunity pathways and substantial differences between the strains beyond their clinical manifestation and pathogenicity. These analyses shed new light into the molecular signature of liver cells upon EBOV infection and reveal new insights into miRNA-based virus attack and host defense strategy.

Whole genome sequence of Corynebacterium kroppenstedtii isolated from a case of recurrent granulomatous mastitis.

We describe the case of a 33-year-old woman with recurrent granulomatous mastitis associated with Corynebacterium kroppenstedtii. This organism has been increasingly associated with granulomatous mastitis, specifically the cystic neutrophilic histopathologic variant, although currently there is a paucity both of reported cases and genomic sequence data. We highlight the challenges in the diagnosis and treatment of this entity, in particular focusing on the various methods of microbiologic identification, including MALDI-TOF, 16 s rRNA PCR and whole-genome sequencing.

Dual RNA-Seq characterization of host and pathogen gene expression in liver cells infected with Crimean-Congo Hemorrhagic Fever Virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that can cause a hemorrhagic fever in humans, with a case fatality rate of up to 40%. Cases of CCHFV have been reported in Africa, Asia, and southern Europe; and recently, due to the expanding range of its vector, autochthonous cases have been reported in Spain. Although it was discovered over 70 years ago, our understanding of the pathogenesis of this virus remains limited. We used RNA-Seq in two human liver cell lines (HepG2 and Huh7) infected with CCHFV (strain IbAr10200), to examine kinetic changes in host expression and viral replication simultaneously at 1 and 3 days post infection. Through this, numerous host pathways were identified that were modulated by the virus including: antiviral response and endothelial cell leakage. Notably, the genes encoding DDX60, a cytosolic component of the RIG-I signalling pathway and OAS2 were both shown to be dysregulated. Interestingly, PTPRR was induced in Huh7 cells but not HepG2 cells. This has been associated with the TLR9 signalling cascade, and polymorphisms in TLR9 have been associated with poor outcomes in patients. Additionally, we performed whole-genome sequencing on CCHFV to assess viral diversity over time, and its relationship to the host response. As a result, we have demonstrated that through next-generation mRNA deep-sequencing it is possible to not only examine mRNA gene expression, but also to examine viral quasispecies and typing of the infecting strain. This demonstrates a proof-of-principle that CCHFV specimens can be analyzed to identify both the virus and host biomarkers that may have implications for prognosis.

Severity of coronavirus respiratory tract infections in adults admitted to acute care in Toronto, Ontario.

BACKGROUND: The World Health Organization has highlighted the need for improved surveillance and understanding of the health burden imposed by non-influenza RNA respiratory viruses. Human coronaviruses (CoVs) are a major cause of respiratory and gastrointestinal tract infections with associated morbidity and mortality. OBJECTIVES: The objective of our study was to characterize the epidemiology of CoVs in our tertiary care centre, and identify clinical correlates of disease severity. STUDY DESIGN: A cross-sectional study was performed of 226 patients admitted with confirmed CoV respiratory tract infection between 2010 and 2016. Variables consistent with a severe disease burden were evaluated including symptoms, length of stay, intensive care unit (ICU) admission and mortality. RESULTS: CoVs represented 11.3% of all positive respiratory virus samples and OC43 was the most commonly identified CoV. The majority of infections were community-associated while 21.6% were considered nosocomial. The average length of stay was 11.8 days with 17.3% of patients requiring ICU admission and an all-cause mortality of 7%. In a multivariate model, female gender and smoking were associated with increased likelihood of admission to ICU or death. CONCLUSION: This study highlights the significant burden of CoVs and justifies the need for surveillance in the acute care setting.

Ebola virus requires phosphatidylinositol (3,5) bisphosphate production for efficient viral entry.

For entry, Ebola virus (EBOV) requires the interaction of its viral glycoprotein with the cellular protein Niemann-Pick C1 (NPC1) which resides in late endosomes and lysosomes. How EBOV is trafficked and delivered to NPC1 and whether this is positively regulated during entry remain unclear. Here, we show that the PIKfyve-ArPIKfyve-Sac3 cellular complex, which is involved in the metabolism of phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P2), is critical for EBOV infection. Although the expression of all subunits of the complex was required for efficient entry, PIKfyve kinase activity was specifically critical for entry by all pathogenic filoviruses. Inhibition of PIKfyve prevented colocalization of EBOV with NPC1 and led to virus accumulation in intracellular vesicles with characteristics of early endosomes. Importantly, genetically-encoded phosphoinositide probes revealed an increase in PtdIns(3,5)P2-positive vesicles in cells during EBOV entry. Taken together, our studies suggest that EBOV requires PtdIns(3,5)P2 production in cells to promote efficient delivery to NPC1.

Replication and Oncolytic Activity of an Avian Orthoreovirus in Human Hepatocellular Carcinoma Cells.

Oncolytic viruses are cancer therapeutics with promising outcomes in pre-clinical and clinical settings. Animal viruses have the possibility to avoid pre-existing immunity in humans, while being safe and immunostimulatory. We isolated an avian orthoreovirus (ARV-PB1), and tested it against a panel of hepatocellular carcinoma cells. We found that ARV-PB1 replicated well and induced strong cytopathic effects. It was determined that one mechanism of cell death was through syncytia formation, resulting in apoptosis and induction of interferon stimulated genes (ISGs). As hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma worldwide, we investigated the effect of ARV-PB1 against cells already infected with this virus. Both HCV replicon-containing and infected cells supported ARV-PB1 replication and underwent cytolysis. Finally, we generated in silico models to compare the structures of human reovirus- and ARV-PB1-derived S1 proteins, which are the primary targets of neutralizing antibodies. Tertiary alignments confirmed that ARV-PB1 differs from its human homolog, suggesting that immunity to human reoviruses would not be a barrier to its use. Therefore, ARV-PB1 can potentially expand the repertoire of oncolytic viruses for treatment of human hepatocellular carcinoma and other malignancies.

Molecular characterization of pathogenic and nonpathogenic fowl aviadenovirus serotype 11 isolates.

Fowl aviadenoviruses, many of which are of importance in veterinary medicine, are classified into 5 species. In this study, a pathogenic isolate and a nonpathogenic isolate of fowl aviadenovirus serotype 11 (FAdV-11) of species Fowl aviadenovirus D were characterized. Growth rates were analyzed for the 2 isolates, showing notable differences. The complete genomic sequences of the viruses were fully determined and were analyzed. The genomes of the 2 isolates showed 98.1% sequence identity and revealed 6 nonsynonymous mutations between the Ontario isolates. Two of the 6 mutations were also found in the sequences of recently published pathogenic Chinese fowl aviadenovirus 11 isolates, suggesting potential molecular markers that could be associated with pathogenesis. Deletions were found in the L5 region within the overlapping coding sequences for the 100, 22, and 33 kDa proteins, and these were found in only the nonpathogenic isolates. This molecular pattern was identified in FAdV-9, another nonpathogenic FAdV-D species virus. Furthermore, the tandem repeat regions varied dramatically; the pathogenic isolates contained a reduced number of tandem repeats compared with the nonpathogenic isolates. Lastly, a protein produced early in infection was analyzed using bioinformatics to determine its role in disease. This study highlights several candidate molecular determinants of avian adenovirus genomes related to pathogenicity.

Characterization of a novel adenovirus isolated from a skunk.

Adenoviruses are a ubiquitous group of viruses that have been found in a wide range of hosts. A novel adenovirus from a skunk suffering from acute hepatitis was isolated and its DNA genome sequenced. The analysis revealed this virus to be a new member of the genus Mastadenovirus, with a genome of 31,848 bp in length containing 30 genes predicted to encode proteins, and with a G+C content of 49.0%. Global genomic organization indicated SkAdV-1 was similar in organization to bat and canine adenoviruses, and phylogenetic comparison suggested these viruses shared a common ancestor. SkAdV-1 demonstrated an ability to replicate in several mammalian liver cell lines suggesting a potential tropism for this virus.

Region of difference 2 contributes to virulence of Mycobacterium tuberculosis.

Mycobacterium bovis BCG strains are live, attenuated vaccines generated through decades of in vitro passage. Because in vitro growth does not select for interaction with the host, it has been hypothesized that genetic loci lost from BCG code for virulence determinants that are dispensable for growth in the laboratory, as exemplified by Region of Difference 1 (RD1), which was lost during the original derivation of BCG between 1908 and 1921. Region of Difference 2 (RD2) was lost during the ongoing propagation of BCG between 1927 and 1931, a time that coincides with reports of the ongoing attenuation of the vaccine. In this study, RD2 has been disrupted in M. tuberculosis H37Rv to test whether its loss contributed to the further attenuation of BCG. The deletion of RD2 did not affect in vitro growth; in contrast, the mutant manifested a decrease in pulmonary and splenic bacterial burdens and reduced pathology in C57BL/6 mice at early time points. This attenuated phenotype was complemented by reintroducing the genes Rv1979c to Rv1982 (including mpt64) but not Rv1985c to Rv1986. In RAW 264.7 macrophages, H37Rv:ΔRD2 showed a decreased proliferation and impaired modulation of the host innate immune response; both observations were complemented with Rv1979c to Rv1982. To test the effect of RD2 disruption on innate immunity, Rag(-/-) mice were infected; H37Rv:ΔRD2 had increased survival times compared those of H37Rv. These findings support the notion that the safety profile of certain BCG vaccines stems from multiple attenuating mutations, with the RD2 deletion resulting in a less-virulent organism through the impaired bacterial manipulation of the host innate immune response.