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BackgroundIn the context of the SARS-CoV-2 pandemic, reuse of personal protective equipment, specifically that of medical face coverings, has been recommended. The reuse of these typically single-use only items necessitates procedures to inactivate contaminating human respiratory and gastrointestinal pathogens. We previously demonstrated decontamination of surgical masks and respirators contaminated with infectious SARS-CoV-2 and various animal coronaviruses via low concentration- and short exposure methylene blue photochemical treatment (10 µM methylene blue, 30 minutes of 12,500-lux red light or 50,000 lux white light exposure).MethodsHere, we describe the adaptation of this protocol to the decontamination of a more resistant, non-enveloped gastrointestinal virus and demonstrate efficient photodynamic inactivation of murine norovirus, a human norovirus surrogate.ResultsMethylene blue photochemical treatment (100 µM methylene blue, 30 minutes of 12,500-lux red light exposure) of murine norovirus-contaminated masks reduced infectious viral titres by over four orders of magnitude on surgical mask surfaces.Discussion and ConclusionsInactivation of a norovirus, the most difficult to inactivate of the respiratory and gastrointestinal human viruses, can predict the inactivation of any less resistant viral mask contaminant. The protocol developed here thus solidifies the position of methylene blue photochemical decontamination as an important tool in the package of practical pandemic preparedness.

To monitor the burden and changes in Haemophilus influenzae (Hi) disease, direct real-time PCR (drt-PCR) assays have been developed for Hi detection in monoplex form and its six serotypes in triplex form, directly from cerebrospinal fluid (CSF) specimens. These assays target the phoB gene for the species detection (Hi-phoB) and serotype-specific genes in region II of the capsule biosynthesis locus (Hi-abf and Hi-cde), identified through comparative analysis of Hi and non-Hi whole-genome sequences. The lower limit of detection (LLD) is 293 CFU/mL for the Hi-phoB assay and ranged from 11 to 130 CFU/mL for the triplex serotyping assays. Using culture as a reference method, the sensitivity and specificity of Hi-phoB, Hi-abf, and Hi-cde were 100%. Triplex serotyping assays also showed 100% agreement for each serotype compared to their corresponding monoplex serotyping assay. These highly sensitive and specific drt-PCR assays do not require DNA extraction and thereby reduce the time, cost, and handling required to process CSF specimens. Furthermore, triplex drt-PCR assays combine the detection of three serotypes in a single reaction, further improving testing efficiency, which is critical for laboratories that process high volumes of Hi specimens for surveillance and diagnostic purposes.

On December 19, 2019, the Food and Drug Administration (FDA) approved rVSVΔG-ZEBOV-GP Ebola vaccine (ERVEBO, Merck) for the prevention of Ebola virus disease (EVD) caused by infection with Ebola virus, species Zaire ebolavirus, in adults aged ≥18 years. In February 2020, the Advisory Committee on Immunization Practices (ACIP) recommended preexposure vaccination with ERVEBO for adults aged ≥18 years in the United States who are at highest risk for potential occupational exposure to Ebola virus because they are responding to an outbreak of EVD, work as health care personnel at federally designated Ebola treatment centers in the United States, or work as laboratorians or other staff members at biosafety level 4 facilities in the United States (1).

Objective:The coronavirus disease 2019 (COVID-19) pandemic has resulted in shortages of personal protective equipment (PPE), underscoring the urgent need for simple, efficient, and inexpensive methods to decontaminate masks and respirators exposed to severe acute respiratory coronavirus virus 2 (SARS-CoV-2). We hypothesized that methylene blue (MB) photochemical treatment, which has various clinical applications, could decontaminate PPE contaminated with coronavirus.Design:The 2 arms of the study included (1) PPE inoculation with coronaviruses followed by MB with light (MBL) decontamination treatment and (2) PPE treatment with MBL for 5 cycles of decontamination to determine maintenance of PPE performance.Methods:MBL treatment was used to inactivate coronaviruses on 3 N95 filtering facepiece respirator (FFR) and 2 medical mask models. We inoculated FFR and medical mask materials with 3 coronaviruses, including SARS-CoV-2, and we treated them with 10 µM MB and exposed them to 50,000 lux of white light or 12,500 lux of red light for 30 minutes. In parallel, integrity was assessed after 5 cycles of decontamination using multiple US and international test methods, and the process was compared with the FDA-authorized vaporized hydrogen peroxide plus ozone (VHP+O3) decontamination method.Results:Overall, MBL robustly and consistently inactivated all 3 coronaviruses with 99.8% to >99.9% virus inactivation across all FFRs and medical masks tested. FFR and medical mask integrity was maintained after 5 cycles of MBL treatment, whereas 1 FFR model failed after 5 cycles of VHP+O3.Conclusions:MBL treatment decontaminated respirators and masks by inactivating 3 tested coronaviruses without compromising integrity through 5 cycles of decontamination. MBL decontamination is effective, is low cost, and does not require specialized equipment, making it applicable in low- to high-resource settings.

The need for increased testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), has resulted in an increase of testing facilities outside of traditional clinical settings and sample handling by individuals without appropriate biohazard and biocontainment training. During the repatriation and quarantine of passengers from the Grand Princess cruise ship at a US military base, biocontainment of a potentially infectious sample from a passenger was compromised. This study describes the steps taken to contain the spill, decontaminate the area, and discusses the needs for adequate training in a biohazard response.

Summary This report summarizes the recommendations of the Advisory Committee on Immunization Practices (ACIP) for use of the rVSVΔG-ZEBOV-GP Ebola vaccine (Ervebo) in the United States. The vaccine contains rice-derived recombinant human serum albumin and live attenuated recombinant vesicular stomatitis virus (VSV) in which the gene encoding the glycoprotein of VSV was replaced with the gene encoding the glycoprotein of Ebola virus species Zaire ebolavirus. Persons with a history of severe allergic reaction (e.g., anaphylaxis) to rice protein should not receive Ervebo. This is the first and only vaccine currently licensed by the Food and Drug Administration for the prevention of Ebola virus disease (EVD). These guidelines will be updated based on availability of new data or as new vaccines are licensed to protect against EVD. ACIP recommends preexposure vaccination with Ervebo for adults aged ≥18 years in the U.S. population who are at highest risk for potential occupational exposure to Ebola virus species Zaire ebolavirus because they are responding to an outbreak of EVD, work as health care personnel at federally designated Ebola treatment centers in the United States, or work as laboratorians or other staff at biosafety level 4 facilities in the United States. Recommendations for use of Ervebo in additional populations at risk for exposure and other settings will be considered and discussed by ACIP in the future.

BackgroundThe coronavirus disease 2019 (COVID-19) pandemic has resulted in severe shortages of personal protective equipment (PPE) necessary to protect front-line healthcare personnel. These shortages underscore the urgent need for simple, efficient, and inexpensive methods to decontaminate SARS-CoV-2-exposed PPE enabling safe reuse of masks and respirators. Efficient decontamination must be available not only in low-resourced settings, but also in well-resourced settings affected by PPE shortages. Methylene blue (MB) photochemical treatment, hitherto with many clinical applications including those used to inactivate virus in plasma, presents a novel approach for widely applicable PPE decontamination. Dry heat (DH) treatment is another potential low-cost decontamination method.MethodsMB and light (MBL) and DH treatments were used to inactivate coronavirus on respirator and mask material. We tested three N95 filtering facepiece respirators (FFRs), two medical masks (MMs), and one cloth community mask (CM). FFR/MM/CM materials were inoculated with SARS-CoV-2 (a Betacoronavirus), murine hepatitis virus (MHV) (a Betacoronavirus), or porcine respiratory coronavirus (PRCV) (an Alphacoronavirus), and treated with 10 µM MB followed by 50,000 lux of broad-spectrum light or 12,500 lux of red light for 30 minutes, or with 75°C DH for 60 minutes. In parallel, we tested respirator and mask integrity using several standard methods and compared to the FDA-authorized vaporized hydrogen peroxide plus ozone (VHP+O3) decontamination method. Intact FFRs/MMs/CM were subjected to five cycles of decontamination (5CD) to assess integrity using International Standardization Organization (ISO), American Society for Testing and Materials (ASTM) International, National Institute for Occupational Safety and Health (NIOSH), and Occupational Safety and Health Administration (OSHA) test methods.FindingsOverall, MBL robustly and consistently inactivated all three coronaviruses with at least a 4-log reduction. DH yielded similar results, with the exception of MHV, which was only reduced by 2-log after treatment. FFR/MM integrity was maintained for 5 cycles of MBL or DH treatment, whereas one FFR failed after 5 cycles of VHP+O3. Baseline performance for the CM was variable, but reduction of integrity was minimal.InterpretationMethylene blue with light and DH treatment decontaminated masks and respirators by inactivating three tested coronaviruses without compromising integrity through 5CD. MBL decontamination of masks is effective, low-cost and does not require specialized equipment, making it applicable in all-resource settings. These attractive features support the utilization and continued development of this novel PPE decontamination method.

During outbreaks of infectious diseases or in cases of severely ill patients, it is imperative to identify the causative agent. This report describes several events in which virus isolation and identification by electron microscopy were critical to initial recognition of the etiologic agent, which was further analyzed by additional laboratory diagnostic assays. Examples include severe acute respiratory syndrome coronavirus, and Nipah, lymphocytic choriomeningitis, West Nile, Cache Valley, and Heartland viruses. These cases illustrate the importance of the techniques of cell culture and electron microscopy in pathogen identification and recognition of emerging diseases.

In 2009, in the Active Bacterial Core surveillance sites, penicillin was not commonly used to treat meningococcal disease. This is likely because of inconsistent availability of antimicrobial susceptibility testing and ease of use of third-generation cephalosporins. Consideration of current practices may inform future meningococcal disease management guidelines.

Containment strategies for outbreaks of invasive Neisseria meningitidis disease are informed by serogroup assays that characterize the polysaccharide capsule. We sought to uncover the genomic basis of conflicting serogroup assay results for an isolate (M16917) from a patient with acute meningococcal disease. To this end, we characterized the complete genome sequence of the M16917 isolate and performed a variety of comparative sequence analyses against N. meningitidis reference genome sequences of known serogroups. Multilocus sequence typing and whole-genome sequence comparison revealed that M16917 is a member of the ST-11 sequence group, which is most often associated with serogroup C. However, sequence similarity comparisons and phylogenetic analysis showed that the serogroup diagnostic capsule polymerase gene ( synD ) of M16917 belongs to serogroup B. These results suggest that a capsule-switching event occurred based on homologous recombination at or around the capsule locus of M16917. Detailed analysis of this locus uncovered the locations of recombination breakpoints in the M16917 genome sequence, which led to the introduction of an ∼2-kb serogroup B sequence cassette into the serogroup C genomic background. Since there is no currently available vaccine for serogroup B strains of N. meningitidis , this kind capsule-switching event could have public health relevance as a vaccine escape mutant.

Background Understanding transmissibility of influenza viruses within households is critical for guiding public health response to pandemics. We studied serologically confirmed infection and disease among household contacts of index case patients with 2009 pandemic influenza A (H1N1) virus (pH1N1) infection in a setting of minimal community pH1N1 transmission. Methods We defined index case patients as students and staff of a New York City high school with laboratory-confirmed pH1N1 infection during the earliest phase of the pH1N1 outbreak in April 2009. We visited households of index case patients twice, once in early May and again in June/July 2009. At each visit, household members (both index case patents and household contacts) provided serum samples and completed questionnaires about illness and possible risk factors. Serologic testing was performed using microneutralization and hemagglutination-inhibition assays. Results Of 79 eligible household contacts in 28 households, 19% had serologically confirmed pH1N1 infection, and 28% of those infected were asymptomatic. Serologically confirmed infection varied by age among household contacts: 36% of contacts younger than 10 years were infected, compared with 46% of contacts age 10-18 years, 8% of contacts aged 19-54 years, and 22% of contacts aged 55 years and older. Conclusions Infection rates were high for household contacts of persons with confirmed pH1N1, particularly for contacts aged 10-18 years, and asymptomatic infection was common. Efforts to reduce household transmission during influenza pandemics are important adjuncts to strategies to reduce community illness.

Since the implementation of Haemophilus influenzae (Hi) serotype b vaccine, other serotypes and non-typeable strains have taken on greater importance as a cause of Hi diseases. A rapid and accurate method is needed to detect all Hi regardless of the encapsulation status. We developed 2 real-time PCR (rt-PCR) assays to detect specific regions of the protein D gene (hpd). Both hpd assays are very specific and sensitive for detection of Hi. Of the 63 non-Hi isolates representing 21 bacterial species, none was detected by the hpd #1 assay, and only one of 2 H. aphrophilus isolates was detected by the hpd #3 assay. The hpd #1 and #3 assays detected 97% (229/237) and 99% (234/237) of Hi isolates, respectively, and were superior for detection of both typeable and non-typeable Hi isolates, as compared to previously developed rt-PCR targeting ompP2 or bexA. The diagnostic sensitivity and specificity of these rt-PCR assays were assessed on cerebrospinal fluid specimens collected as part of meningitis surveillance in Ulaanbaatar, Mongolia. The etiology (Neisseria meningitidis, Hi, and Streptococcus pneumoniae) of 111 suspected meningitis cases was determined by conventional methods (culture and latex agglutination), previously developed rt-PCR assays, and the new hpd assays. The rt-PCR assays were more sensitive for detection of meningitis pathogens than other classical methods and improved detection from 50% (56/111) to 75% (83/111). The hpd #3 assay identified a non-b Hi that was missed by the bexA assay and other methods. A sensitive rt-PCR assay to detect both typeable and non-typeable Hi is a useful tool for improving Hi disease surveillance especially after Hib vaccine introduction.

Background. A quadrivalent meningococcal conjugate vaccine (MCV4) was licensed in the United States in 2005; no serogroup B vaccine is available. Neisseria meningitidis changes its capsular phenotype through capsular switching, which has implications for vaccines that do not protect against all serogroups. Methods. Meningococcal isolates from 10 Active Bacterial Core surveillance sites from 2000 through 2005 were analyzed to identify changes occurring after MCV4 licensure. Isolates were characterized by multilocus sequence typing (MLST) and outer membrane protein gene sequencing. Isolates expressing capsular polysaccharide different from that associated with the MLST lineage were considered to demonstrate capsular switching. Results. Among 1160 isolates, the most common genetic lineages were the sequence type (ST)-23, ST-32, ST-11, and ST-41/44 clonal complexes. Of serogroup B and Y isolates, 8 (1.5%) and 3 (0.9%), respectively, demonstrated capsular switching, compared with 36 (12.9%) for serogroup C (P

Nipah virus (NiV) and Hendra virus (HeV) are zoonotic paramyxoviruses capable of causing severe disease in humans and animals. These viruses require biosafety level 4 (BSL-4) containment. Like other paramyxoviruses, the plaque reduction neutralization test (PRNT) can be used to detect antibodies to the surface glycoproteins, fusion (F) and attachment (G), and PRNT titers give an indication of protective immunity. Unfortunately, for NiV and HeV, the PRNT must be performed in BSL-4 containment and takes several days to complete. Thus, we have developed a neutralization assay using VSV pseudotype particles expressing the F and G proteins of NiV (pVSV-NiV-F/G) as target antigens. This rapid assay, which can be performed at BSL-2, was evaluated using serum samples from outbreak investigations and more than 300 serum samples from an experimental NiV vaccination study in swine. The results of the neutralization assays with pVSV-NiV-F/G as antigen showed a good correlation with those of standard PRNT. Therefore, this new method has the potential to be a rapid and cost-effective diagnostic method, especially in locations that lack high containment facilities, and will provide a valuable tool for basic research and vaccine development.

We report on three cases of meningococcal disease caused by ciprofloxacin-resistant Neisseria meningitidis, one in North Dakota and two in Minnesota. The cases were caused by the same serogroup B strain. To assess local carriage of resistant N. meningitidis, we conducted a pharyngeal-carriage survey and isolated the resistant strain from one asymptomatic carrier. Sequencing of the gene encoding subunit A of DNA gyrase (gyrA) revealed a mutation associated with fluoroquinolone resistance and suggests that the resistance was acquired by means of horizontal gene transfer with the commensal N. lactamica. In susceptibility testing of invasive N. meningitidis isolates from the Active Bacterial Core surveillance system between January 2007 and January 2008, an additional ciprofloxacin-resistant isolate was found, in this case from California. Ciprofloxacin-resistant N. meningitidis has emerged in North America.

Isolates of Neisseria meningitidis collected in 2004, 2008, 2010, and 2011 from an active population-based surveillance system in the United States were tested for susceptibility to antimicrobials used for treatment and chemoprophylaxis of meningococcal disease. Susceptibility remains high and stable., Background. Antimicrobial treatment and chemoprophylaxis of patients and their close contacts is critical to reduce the morbidity and mortality and prevent secondary cases of meningococcal disease. Through the 1990's, the prevalence of antimicrobial resistance to commonly used antimicrobials among Neisseria meningitidis was low in the United States. Susceptibility testing was performed to ascertain whether the proportions of isolates with reduced susceptibility to antimicrobials commonly used for N meningitidis have increased since 2004 in the United States. Methods. Antimicrobial susceptibility testing was performed by broth microdilution on 466 isolates of N meningitidis collected in 2004, 2008, 2010, and 2011 from an active, population-based surveillance system for susceptibility to ceftriaxone, ciprofloxacin, penicillin G, rifampin, and azithromycin. The molecular mechanism of reduced susceptibility was investigated for isolates with intermediate or resistant phenotypes. Results. All isolates were susceptible to ceftriaxone and azithromycin, 10.3% were penicillin G intermediate (range, 8% in 2008–16.7% in 2010), and

Design, synthesis, and biological evaluation of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease (SARS-3CLpro) inhibitors for severe acute respiratory syndrome coronavirus (SARS-CoV) are described. These inhibitors exhibited antiviral activity against SARS-CoV in infected cells in the micromolar range. An X-ray crystal structure of our lead inhibitor (4) bound to SARS-3CLpro provided important drug-design templates for the design of small-molecule inhibitors.

Monoclonal antibodies (Mabs) against the Urbani strain of the SARS-associated coronavirus (SARS-CoV) were developed and characterized for reactivity to SARS-CoV and SARS-CoV S, N, M, and E proteins using enzyme-linked immunoabsorbent (ELISA), radioimmunoprecipitation, immunofluorescence, Western Blot and microneutralization assays. Twenty-six mAbs were reactive to SARS-CoV by ELISA, and nine were chosen for detailed characterization. Five mAbs reacted against the S protein, two against the M protein, and one each against the N and E proteins. Two of five S protein mAbs neutralized SARS-CoV infection of Vero E6 cells and reacted to an epitope within amino acids 490-510 in the S protein. While two of the three non-neutralizing antibodies recognized at second epitope within amino acids 270-350. The mAbs characterized should prove useful for developing SARS-CoV diagnostic assays and for studying the biology of infection and pathogenesis of disease.

Nipah virus is a recently emergent paramyxovirus that is capable of causing severe disease in both humans and animals. The first outbreak of Nipah virus occurred in Malaysia and Singapore in 1999 and, more recently, outbreaks were detected in Bangladesh. In humans, Nipah virus causes febrile encephalitis with respiratory syndrome that has a high mortality rate. The reservoir for Nipah virus is believed to be fruit bats, and humans are infected by contact with infected bats or by contact with an intermediate animal host such as pigs. Person to person spread of the virus has also been described. Nipah virus retains many of the genetic and biologic properties found in other paramyxoviruses, though it also has several unique characteristics. However, the virologic characteristics that allow the virus to cause severe disease over a broad host range, and the epidemiologic, environmental and virologic features that favor transmission to humans are unknown. This review summarizes what is known about the virology, epidemiology, pathology, diagnosis and control of this novel pathogen.

A novel coronavirus (CoV) has been described in association with cases of severe acute respiratory syndrome (SARS). The virus, SARS-CoV, differs from the previously described human coronaviruses, 229E and OC43. 229E was previously shown to productively infect human monocytes/macrophages, whereas OC43 poorly infected the cells. In this study, we examined whether SARS-CoV could productively infect purified monocytes/macrophages (PM) derived from human donor cells. Unlike 229E-infected cells, which produced viral titers of 10(3.5) to 10(6)TCID50/ml, SARS-CoV replicated poorly in PM, producing titers of 10(1.75) to 10(2)TCID50/ml. This finding was similar to results reported for OC43-infected cells, with titers ranging from 10(1.2) to 10(2.7)TCID50/ml. Of interest, SARS-CoV proteins were detected only in PM that did not produce significant amounts of interferon (IFN)-alpha, and in one such case, preliminary electron microscope studies demonstrated that SARS-CoV-like particles could enter the cells, possibly via phagocytosis. These results suggest that SARS-CoV, like human CoV OC43, poorly infects human PM, and production of IFN-alpha by these cells further limits the infection. Given the importance of monocytes/macrophages to the immune response, it is possible that their infection by SARS-CoV and alteration of this infection by IFN-alpha may be important to the course of the infection in humans.

Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nsp1, nsp2, and nsp3 and describe trans -cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S -transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nsp1 (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a ≈300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans -cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nsp1/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.

To study the replication of Nipah virus (NiV), a minigenome replication assay that does not require the use of infectious virus was developed. The minigenome was constructed to encode a NiV vRNA analogue containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative NiV transcription motifs and flanked by the NiV genomic termini. CAT protein was detected only when plasmids encoding the NiV minigenome, nucleocapsid protein (N), phosphoprotein (P) and polymerase protein (L) were transfected into CV1 cells. To determine whether NiV conforms to the rule of six, a series of plasmids encoding minigenomes that differed in length by a single nucleotide was tested in the replication assay. CAT production was detected only with the minigenome whose length was an even multiple of six. The replication assay was also used to show that the N, P and L proteins of NiV recognize cis-acting sequences in the genomic termini of Hendra virus (HeV) but not measles virus. While these results suggest that NiV uses a replication strategy that is similar to those of other paramyxoviruses, they also support the inclusion of NiV and HeV in a separate genus within the subfamily Paramyxovirinae.

In 1998, Nipah virus (NV) emerged in peninsular Malaysia, causing fatal encephalitis in humans and a respiratory disease in swine. NV is most closely related to Hendra virus (HV), a paramyxovirus that was identified in Australia in 1994, and it has been proposed that HV and NV represent a new genus within the family Paramyxoviridae. This report describes the analysis of the sequences of the polymerase gene (L) and genomic termini of NV as well as a comparison of the full-length, genomic sequences of HV and NV. The L gene of NV is predicted to be 2244 amino acids in size and contains the six domains found within the L proteins of all nonsegmented, negative-stranded (NNS) RNA viruses. However, the GDNQ motif found in most NNS RNA viruses was replaced by GDNE in both NV and HV. The 3′ and 5′ termini of the NV genome are nearly identical to the genomic termini of HV and share sequence homology with the genomic termini of other members of the subfamily Paramyxovirinae. At 18,246 nucleotides, the genome of NV is 12 nucleotides longer than the genome of HV and they have the largest genomes within the family Paramyxoviridae. The comparison of the structures of the genomes of HV and NV is now complete and this information will help to establish the taxonomic position of these novel viruses within the family Paramyxoviridae.

The structure and genetic organization of Hendra and Nipah viruses places them in the subfamily Paramyxovirinae. However, low homology with other subfamily members and several novel biological and molecular features such as genome length and F(0 )cleavage site suggest classification in a new genus within the Paramyxovirinae.

The expression of intercellular adhesion molecule 1 (ICAM-1) by endothelial cells is important for the regulation of adhesion and transendothelial migration of a variety of leukocytes that express the integrins lymphocyte function-associated antigen 1 (LFA-1) and/or Mac-1. Here, we demonstrate strain-specific differences in the ability of measles virus (MV) to induce ICAM-1 expression. The vaccine strain Moraten (Mor) rapidly induced high levels of ICAM-1 mRNA and protein expression, whereas the vaccine strain CAM-70 and the Edmonston wild type (Ed-wt) strain were far less effective, even when they were used at very high multiplicities of infection (MOIs). Strain-specific differences in the induction were not a consequence of differences in the ability to infect ECs. Furthermore, induction of ICAM-1 by Mor was not dependent on de novo expression of MV or cellular proteins. Dual-immunofluorescence analysis indicated that there was no association between the expression of either MV nucleocapsid or hemagglutinin protein and the induction of ICAM-1 expression. Some human umbilical vein endothelial cells (HUVECs) that expressed high nucleocapsid protein in response to either Mor or CAM-70 failed to express elevated ICAM-1, whereas some HUVECs that were incubated with Mor expressed high ICAM-1 prior to expression of MV nucleocapsid protein. Strain-specific differences in the ability of Mor and CAM-70 to induce ICAM-1 correlated with their ability to activate the latent transcription factor NF-kappaB. These studies suggest a preexisting component of MV particles that leads to strain-specific differences in the activation of NF-kappaB and the induction of ICAM-1 gene expression.

Fatal cases of filoviral infection are accompanied by a marked immunosuppression. Endothelial cells play a vital role in the host immune response through the expression of several immunomodulatory genes in addition to the expression of the antiviral genes, 2′,5′-oligoadenylate synthetase [2′-5′(A) N ], and the double-stranded RNA (dsRNA)-activated protein kinase (PKR). dsRNA, an intermediate generated during viral replication and gene transcription of many viruses, leads to the induction of immunomodulatory genes in endothelial cells. In this report, we show that induction of the major histocompatibility complex class I family of genes, 2′-5′(A) N , interleukin-6 (IL-6), PKR, interferon (IFN)-regulatory factor-1, and intercellular adhesion molecule-1 (ICAM-1) by dsRNA in human umbilical vein endothelial cells is suppressed by infection with the filovirus Ebola–Zaire (EZ). In contrast, induction of IL-6 and ICAM-1 by IL-1 is intact in EZ-infected cells. Gel shift analysis demonstrates that dsRNA-induced protein binding to IFN-responsive elements is strongly suppressed by EZ-IFN, whereas NF-κB activation by dsRNA remains intact. We previously reported that IFN signaling is suppressed by EZ infection, and these data strongly suggest that elements shared between IFN and dsRNA signaling are being inhibited by EZ. Inhibition of IFN and dsRNA responsiveness could play a role in the immunosuppression seen in EZ infections and would play a role in the pathogenesis of disease caused by EZ.

Background Although cerebrospinal fluid (CSF) culture is the diagnostic reference standard for bacterial meningitis, its sensitivity is limited, particularly when antibiotics were previously administered. CSF Gram staining and real-time PCR are theoretically less affected by antibiotics; however, it is difficult to evaluate these tests with an imperfect reference standard. Methods and findings CSF from patients with suspected meningitis from Salvador, Brazil were tested with culture, Gram stain, and real-time PCR using S. pneumoniae, N. meningitidis, and H. influenzae specific primers and probes. An antibiotic detection disk bioassay was used to test for the presence of antibiotic activity in CSF. The diagnostic accuracy of tests were evaluated using multiple methods, including direct evaluation of Gram stain and real-time PCR against CSF culture, evaluation of real-time PCR against a composite reference standard, and latent class analysis modeling to evaluate all three tests simultaneously. Results Among 451 CSF specimens, 80 (17.7%) had culture isolation of one of the three pathogens (40 S. pneumoniae, 36 N. meningitidis, and 4 H. influenzae), and 113 (25.1%) were real-time PCR positive (51 S. pneumoniae, 57 N. meningitidis, and 5 H. influenzae). Compared to culture, real-time PCR sensitivity and specificity were 95.0% and 90.0%, respectively. In a latent class analysis model, the sensitivity and specificity estimates were: culture, 81.3% and 99.7%; Gram stain, 98.2% and 98.7%; and real-time PCR, 95.7% and 94.3%, respectively. Gram stain and real-time PCR sensitivity did not change significantly when there was antibiotic activity in the CSF. Conclusion Real-time PCR and Gram stain were highly accurate in diagnosing meningitis caused by S. pneumoniae, N. meningitidis, and H. influenzae, though there were few cases of H. influenzae. Furthermore, real-time PCR and Gram staining were less affected by antibiotic presence and might be useful when antibiotics were previously administered. Gram staining, which is inexpensive and commonly available, should be encouraged in all clinical settings.

Neisseria meningitidis is one of the main agents of bacterial meningitis, causing substantial morbidity and mortality worldwide. However, most of the time N. meningitidis is carried as a commensal not associated with invasive disease. The genomic basis of the difference between disease-associated and carried isolates of N. meningitidis may provide critical insight into mechanisms of virulence, yet it has remained elusive. Here, we have taken a comparative genomics approach to interrogate the difference between disease-associated and carried isolates of N. meningitidis at the level of individual nucleotide variations (i.e., single nucleotide polymorphisms [SNPs]). We aligned complete genome sequences of 8 disease-associated and 4 carried isolates of N. meningitidis to search for SNPs that show mutually exclusive patterns of variation between the two groups. We found 63 SNPs that distinguish the 8 disease-associated genomes from the 4 carried genomes of N. meningitidis , which is far more than can be expected by chance alone given the level of nucleotide variation among the genomes. The putative list of SNPs that discriminate between disease-associated and carriage genomes may be expected to change with increased sampling or changes in the identities of the isolates being compared. Nevertheless, we show that these discriminating SNPs are more likely to reflect phenotypic differences than shared evolutionary history. Discriminating SNPs were mapped to genes, and the functions of the genes were evaluated for possible connections to virulence mechanisms. A number of overrepresented functional categories related to virulence were uncovered among SNP-associated genes, including genes related to the category “symbiosis, encompassing mutualism through parasitism.”

Background. An outbreak of serogroup C meningococcal disease that involved illicit drug users and their contacts occurred in Brooklyn, New York, during 2005 and 2006. Methods. The objectives of this study were to identify the population at risk for meningococcal disease, describe efforts to interrupt disease transmission, and assess the impact of a vaccine initiative. Descriptive and molecular epidemiological analysis was used to define the extent of the outbreak and the common risk factors among outbreak-related cases. A vaccine initiative that used community-based service providers was targeted to illicit drug users and their close contacts. The vaccine initiative was assessed through cessation of outbreak-related cases and the reduction in carriage rate. Results. The investigation identified 23 outbreak-related cases of serogroup C meningococcal disease; 17 isolates were indistinguishable and 4 isolates were closely related according to pulsed-field gel electrophoresis. Two additional culture-negative cases had epidemiological links to laboratory-confirmed cases. The median age of patients with outbreak-related cases was 41 years, and 19 (83%) of 23 patients reported an association with illicit drug use. There were 7 outbreak-related deaths. Vaccination was administered to 2763 persons at 29 community locations, including methadone treatment centers, syringe-exchange programs, and soup kitchens. Three additional cases of meningococcal disease due to strains with the same pulsed-field gel electrophoresis pattern were identified after the vaccination initiative. Conclusions. Community-based outbreaks of meningococcal disease are difficult to control, and the decision to vaccinate is not straightforward. Current national guidelines for implementing a vaccination campaign are not strict criteria and cannot be expected to accommodate the myriad of factors that occur in community-based invasive meningococcal disease outbreaks, such as the inability to enumerate the population at risk.

The henipaviruses, Nipah virus (NiV) and Hendra virus (HeV), are highly pathogenic zoonotic paramyxoviruses. Like many other paramyxoviruses, henipaviruses employ a process of co-transcriptional mRNA editing during transcription of the phosphoprotein (P) gene to generate additional mRNAs encoding the V and W proteins. The C protein is translated from the P mRNA, but in an alternate reading frame. Sequence analysis of multiple, cloned mRNAs showed that the mRNA editing frequencies of the P genes of the henipaviruses are higher than those reported for other paramyxoviruses. Antisera to synthetic peptides from the P, V, W and C proteins of NiV were generated to study their expression in infected cells. All proteins were detected in both infected cells and purified virions. In infected cells, the W protein was detected in the nucleus while P, V and C were found in the cytoplasm.

Until 2004, identification of Nipah virus (NV)-like outbreaks in Bangladesh was based on serology. We describe the genetic characterization of a new strain of NV isolated during outbreaks in Bangladesh (NV-B) in 2004, which confirms that NV was the etiologic agent responsible for these outbreaks.

Nipah virus (NV) and Hendra virus (HV) are recently emergent, related viruses that can cause severe disease in humans and animals. The goal of this study was to investigate the immunogenic and functional properties of the fusion (F) and attachment (G) glycoproteins of NV. Vaccination of mice with recombinant vaccinia viruses (rVVs) expressing either the F (rVV/NV-F) or G (rVV/NV-G) proteins of NV induced neutralizing antibody responses to NV, with higher titers produced after vaccination with rVV/NV-G. When the homologous pairs of F and G proteins from either HV or NV were coexpressed in a transient expression system, fusion was detected in less than 12 h. An equivalent amount of fusion was observed when the heterologous pairs of F and G proteins from HV and NV were coexpressed. Membrane fusion was inhibited by antiserum from mice vaccinated with rVV/NV-G and rVV/NV-F. Therefore, as with other paramyxoviruses, the membrane glycoproteins of NV are the targets of neutralizing antibodies and membrane fusion mediated by NV requires the presence of both the F and the G proteins. Data from these biological assays support the taxonomic grouping of both HV and NV in the new genus, Henipavirus, within the family Paramyxoviridae.

Ebola virus infection is highly lethal and leads to severe immunosuppression. In this study, we demonstrate that infection of human umbilical vein endothelial cells (HUVECs) with Ebola virus Zaire (EZ) suppressed basal expression of the major histocompatibility complex class I (MHC I) family of proteins and inhibited the induction of multiple genes by alpha interferon (IFN-α) and IFN-γ, including those coding for MHC I proteins, 2′-5′ oligoadenylate synthetase [2′-5′(A) N ], and IFN regulatory factor 1 (IRF-1). Induction of interleukin-6 (IL-6) and ICAM-1 by IL-1β was not suppressed by infection with EZ, suggesting that the inhibition of IFN signaling is specific. Gel shift analysis demonstrated that infection with EZ blocked the induction by IFNs of nuclear proteins that bind to IFN-stimulated response elements, gamma activation sequences, and IFN regulatory factor binding site (IRF-E). In contrast, infection with EZ did not block activation of the transcription factor NF-κB by IL-1β. The events that lead to the blockage of IFN signaling may be critical for Ebola virus-induced immunosuppression and would play a role in the pathogenesis of Ebola virus infection.

Recently, a new paramyxovirus, now known as Nipah virus (NV), emerged in Malaysia and Singapore, causing fatal encephalitis in humans and a respiratory syndrome in pigs. Initial studies had indicated that NV is antigenically and genetically related to Hendra virus (HV). We generated the sequences of the N, P/C/V, M, F, and G genes of NV and compared these sequences with those of HV and other members of the family Paramyxoviridae. The intergenic regions of NV were identical to those of HV, and the gene start and stop sequences of NV were nearly identical to those of HV. The open reading frames (ORFs) for the V and C proteins within the P gene were found in NV, but the ORF encoding a potential short basic protein found in the P gene of HV was not conserved in NV. The N, P, C, V, M, F, and G ORFs in NV have nucleotide homologies ranging from 88% to 70% and predicted amino acid homologies ranging from 92% to 67% in comparison with HV. The predicted fusion cleavage sequence of the F protein of NV had a single amino acid substitution (K to R) in comparison with HV. Phylogenetic analysis demonstrated that although HV and NV are closely related, they are clearly distinct from any of the established genera within the Paramyxoviridae and should be considered a new genus.

International audience; In 2015, Niger reported the largest epidemic of Neisseria meningitidis serogroup C (NmC) meningitis in sub-Saharan Africa. The NmC epidemic coincided with serogroup W (NmW) cases during the epidemic season, resulting in a total of 9,367 meningococcal cases through June 2015. To clarify the phylogenetic association, genetic evolution, and antibiotic determinants of the meningococcal strains in Niger, we sequenced the genomes of 102 isolates from this epidemic, comprising 81 NmC and 21 NmW isolates. The genomes of 82 isolates were completed, and all 102 were included in the analysis. All NmC isolates had sequence type 10217, which caused the outbreaks in Nigeria during 2013-2014 and for which a clonal complex has not yet been defined. The NmC isolates from Niger were substantially different from other NmC isolates collected globally. All NmW isolates belonged to clonal complex 11 and were closely related to the isolates causing recent outbreaks in Africa.