Recent Isolates Research Articles

Recent Clinical Isolates of Enterovirus D68 Have Increased Replication and Induce Enhanced Epithelial Immune Response Compared to the Prototype Fermon Strain.

In 2014, enterovirus D68 (EV-D68), previously associated primarily with mild respiratory illness, caused a large outbreak of severe respiratory illness and, in rare instances, paralysis. We compared the viral binding and replication of eight recent EV-D68 clinical isolates collected both before and during the 2014 outbreak and the prototype Fermon strain from 1962 in cultured HeLa cells and differentiated human primary bronchial epithelial cells (BEC) to understand the possible reasons for the change in virus pathogenicity. We selected pairs of closely related isolates from the same phylogenetic clade that were associated with severe vs. asymptomatic infections. We found no significant differences in binding or replication in HeLa cell cultures between the recent clinical isolates. However, in HeLa cells, Fermon had significantly greater binding (2-3 logs) and virus progeny yields (2-4 logs) but a similar level of replication (1.5-2 log increase in viral RNA from 2 h to 24 h post infection) compared to recent isolates. In differentiated BECs, Fermon and the recent EV-D68 isolates had similar levels of binding; however, the recent isolates produced 1.5-2-log higher virus progeny yields than Fermon due to increased replication. Interestingly, no significant differences in replication were identified between the pairs of genetically close recent EV-D68 clinical isolates despite the observed differences in associated disease severity. We then utilized RNA-seq to define the transcriptional responses in BECs infected with four recent EV-D68 isolates, representing major phylogenetic clades, and the Fermon strain. All the tested clinical isolates induced similar responses in BECs; however, numerous upregulated genes in antiviral and pro-inflammatory response pathways were identified when comparing the response to clinical isolates versus Fermon. These results indicate that the recent emergence in severe EV-D68 cases could be explained by an increased replication efficiency and enhanced inflammatory response induced by newly emerged clinical isolates; however, host factors are likely the main determinants of illness severity.

Determination of protection provided by two modified live bovine herpesvirus-1 vaccines against bovine herpesvirus-1 conjunctivitis in calves

A study was performed in 2 phases to determine the ability of 2 different modified live (MLV) bovine herpesvirus-1 (BoHV-1)vaccines to protect against conjunctivitis caused by a recent field isolate of BoHV-1. In phase I, BoHV-1 isolates were used todetermine the ability of the isolates to cause conjunctivitis and to establish a challenge model for phase II. Fifteen calves wererandomly assigned to 1 of 3 groups: 5 calves received 5 mL containing 107 tissue culture infectious dose (TCID)50/mL of BoHV-1adminisered intraocularly (topically) for 2 days, 5 calves received 5 mL of inoculum consisting of 107TCID50/mL of BoHV-1 administered intranasally for 2 days, and 5 calves served as unchallenged sentinels and received phosphatebuffered saline, 5 mL administered by the intranasal and 5 mL by the intraocular route for 2 days. All 15 calves developedpyrexia (rectal temperature greater than 103.5°F (39.7°C)), were BoHV-1 virus isolation positive from ocular swabsand 10/15 calves had increased ocular discharge and conjunctivitis. In phase II, 10 calves were intranasally vaccinated witha MLV BoHV-1, 10 calves were parenterally vaccinated with a MLV BoHV-1, and 5 calves were unvaccinated. All 25 calveswere challenged by receiving 5 mL ntraocularly of 107 TCID50/mL of BoHV-1 for 2 consecutive days. Following challenge, calves were examined daily, and blood (serum), nasal swabs, and conjunctival swab samples were obtained on study days 0, 3-7, 10, 14 and 28. Both vaccines significantly decreased post challenge ocular lesions, clinical scores, and quantity of virus isolated.

Genetic characterization and phylogenetic analysis of foot and mouth disease virus vaccine strains and recent field isolate

Foot and Mouth Disease (FMD) control is a national and regional responsibility. FMD outbreaks were detected in three farms in Port-Said Governorate in 2020, although animals in these farms were vaccinated with the local polyvalent inactivated vaccine. Samples from tongue epithelium and vesicular fluid were collected from these farms, where FMDV isolation was performed on BHK-21 cell culture. Viral RNA was extracted from the virus isolates, and vaccine strains were then screened for FMDV using conventional Reverse transcriptase polymerase chain reaction. The substitution rates and phylogenetic relationship between the field isolate and the vaccine strains were determined using DNA sequence and bioinformatics analyses. The results illustrate that the sequence analysis for the 1D region of FMDV field isolate was serotype A‑Africa topotype, Genotype IV, and closely related to isolated A‑Africa topotype Genotype IV by Animal Health Research Institute, 2020 with nucleotide similarity ranging from 97.1% to 99.74%, and revealing genetic variation of 5.63-7.88% from previously Egyptian isolated A‑Africa topotype, Genotype IV in 2016 and 2018. High genetic variations were determined from the locally used vaccine strain serotype A of the Asia topotype, lineage Iran‑05, in the major antigenic sites of the VP1 region with a nucleotide difference of 26.34%. Depending on these findings, the Veterinary Serum and Vaccine Research Institute produced an emergency inactivated monovalent vaccine using the newly isolated strain, and we recommended adding it to the subsequent prepared vaccine batches.

Development and validation of multiplex PCR based molecular serotyping of Salmonella serovars associated with poultry in India

Salmonella species are Gram-negative bacteria with more than 2600 serovars. Among these serovars, many are associated with various diseases in livestock and humans. White Kauffman Le-Minor (WKL) serotyping scheme applies specific serum to determine the serovars of Salmonella. Recent studies have applied molecular methods for serovar predictions. These methods include PCR, hybridization and sequence data to detect/predict serovar-specific genetic elements. Among these, PCR is a robust method if the unique genetic element is already known. Within this context, also involving novel primers, two multiplex PCR assays were standardized to detect six important Salmonella serovars viz. Typhimurium, Enteritidis, Kentucky, Infantis, Virchow and Gallinarum associated with poultry in India. The developed PCR assays showed targeted serovar specificity. Serial dilution experiments of both kit-based and crude lysate DNA preparations indicated similar applicability of both methods for testing from pure cultures. Further the developed assays were validated with 25 recent field isolates to confirm the applicability in routine diagnosis. The PCR assay could predict all the targeted serovars (17/25) with 100% specificity (CI-95%; 0.63–1). Molecular serotyping can reduce the number of serum used in comparison to the conventional serotyping which involves more random application of serum.

Emergence and genomic analysis of a novel sublineage of bovine ephemeral fever virus in Southwest China.

Bovine ephemeral fever virus (BEFV), belonging to the genus Ephemerovirus under the family Rhabdoviridae, is the etiological cause for the bovine ephemeral fever (BEF) in cattle and water buffalo. In this study, we report recent BEF outbreaks in Southwest China and sequence the complete genome sequence of one BEFV isolate BEFV/CQ1/2022. Comparative genomic analyses between BEFV/CQ1/2022 and isolates available in GenBank revealed remarkable inter-isolate divergence. Meanwhile, the sequence divergence was related to the evolutionary relationships and geographical distribution of the isolates. Phylogenetic analysis indicated that the global BEFV isolates can be divided into 4 distinct lineages. The East Asia lineage was the most diverse and could be subdivided into 4 sublineages. Notably, BEFV/CQ1/2022 and other 10 recent isolates from Mainland China were found to be clustered in sublineage 2. Additionally, recombination analysis provided evidence of BEFV recombination among East Asian isolates for the first time. Taken together, a novel sublineage of the East Asian BEFV emerged in Southwest China, and large divergence and potential recombination among BEFV strains were investigated in this study, which may improve understanding of BEFV epidemiology and evolution.

Comparative pathogenicity of a genotype XXI.1.2 pigeon Newcastle disease virus isolate in pigeons and chickens

Here, we performed molecular and pathogenic characterization of a Newcastle disease virus (NDV) isolate from pigeons in Bangladesh. Molecular phylogenetic analysis based on the complete fusion gene sequences classified the three study isolates into genotype XXI (sub-genotype XXI.1.2) together with recent NDV isolates obtained from pigeons in Pakistan (2014–2018). The Bayesian Markov Chain Monte Carlo analysis revealed that the ancestor of Bangladeshi pigeon NDVs and the viruses from sub-genotype XXI.1.2 existed in the late 1990s. Pathogenicity testing using mean embryo death time pathotyped the viruses as mesogenic, while all isolates carried multiple basic amino acid residues at the fusion protein cleavage site. Experimental infection of chickens and pigeons revealed no or minimum clinical signs in chickens, while a relatively high morbidity (70%) and mortality (60%) were observed in pigeons. The infected pigeons showed extensive and systemic lesions including hemorrhagic and/or vascular changes in the conjunctiva, respiratory and digestive system and brain, and atrophy in the spleen, while only mild congestion in the lungs was noticed in the inoculated chickens. Histologically, consolidation in the lungs with collapsed alveoli and edema around the blood vessels, hemorrhages in the trachea, severe hemorrhages and congestion, focal aggregation of mononuclear cells, and single hepatocellular necrosis in the liver, severe congestion, multifocal tubular degeneration, and necrosis, as well as mononuclear cell infiltration in the renal parenchyma, encephalomalacia with severe neuronal necrosis with neuronophagia were noticed in the brain in infected pigeons. In contrast, only slight congestion was found in lungs of the infected chickens. qRT-PCR revealed the replication of the virus in both pigeons and chickens; however, higher viral RNA loads were observed in oropharyngeal and cloacal swabs, respiratory tissues, and spleen of infected pigeons than the chickens. In conclusion, genotype XXI.1.2 NDVs are circulating in the pigeon population of Bangladesh since 1990s, produce high mortality in pigeons with pneumonia, hepatocellular necrosis, renal tubular degeneration, and neuronal necrosis in pigeons, and may infect chickens without overt signs of clinical disease and are likely to shed viruses via the oral or cloacal routes.

Based on the Results of PEDV Phylogenetic Analysis of the Most Recent Isolates in China, the Occurrence of Further Mutations in the Antigenic Site S1° and COE of the S Protein Which Is the Target Protein of the Vaccine

The continuous challenge of existing vaccine systems by porcine epidemic diarrhea virus (PEDV) variant strains in recent years, it caused significant economic losses in the global swine industry. A PEDV virulent strain CH/HLJBQL/2022 was successfully isolated in China in this study. A genome-wide based phylogenetic analysis suggests that CH/HLJBQL/2022 belongs to the GII subtype, and 96.3%–99.6% homology existed in the whole genomes of other strains. For the first time, simultaneous mutations of four amino acids were found in the highly conserved membrane (M) and nucleocapsid (N) proteins, as well as five amino acid mutations that differed from the vast majority of strains in the spike (S) protein. Mutations in the M and S proteins were found to produce coils with different angles by building 2D and 3D structural models. Epitope analysis indicated that the isolates produced specific changes and that the transmembrane function of the M protein had not been affected. In addition, typing markers exist during strain evolution, but isolates are using the fusion of specific amino acids from multiple variant strains to add additional features, as also demonstrated by protein alignments and 3D models of numerous subtype strains. These results suggest that aa mutations in the M and S proteins may have changed the structure and antigenic epitope of the isolates and PEDV is evolving again on the basis of variants that have been found to counteract the immune network of the new vaccine.

Whole-genome sequence and genesis of an avian influenza virus H5N1 isolated from a healthy chicken in a live bird market in Indonesia: accumulation of mammalian adaptation markers in avian hosts.

Influenza A viruses are a major pathogen that causes significant clinical and economic harm to many animals. In Indonesia, the highly pathogenic avian influenza (HPAI) H5N1 virus has been endemic in poultry since 2003 and has caused sporadic deadly infections in humans. The genetic bases that determine host range have not yet been fully elucidated. We analyzed the whole-genome sequence of a recent H5 isolate to reveal the evolution toward its mammalian adaptation. We determined the whole-genome sequence of A/chicken/East Java/Av1955/2022 (hereafter, "Av1955") from a healthy chicken in April 2022 and conducted phylogenetic and mutational analysis. Phylogenetic analysis revealed that Av1955 belonged to the H5N1 clade 2.3.2.1c (Eurasian lineage). The six gene segments (PB1, PB2, HA, NP, NA, and NS) out of the eight segments derived from viruses of H5N1 Eurasian lineage, one (PB2) from the H3N6 subtype and the remaining one (M) from the H5N1 clade 2.1.3.2b (Indonesian lineage). The donor of the PB2 segment was a reassortant among three viruses of H5N1 Eurasian and Indonesian lineages and the H3N6 subtype. The HA amino acid sequence contained multiple basic amino acids at the cleavage site. Mutation analysis revealed that Av1955 possessed the maximal number of mammalian adaptation marker mutations. Av1955 was a virus of H5N1 Eurasian lineage. The HA protein contains an HPAI H5N1-type cleavage site sequence, while the virus was isolated from a healthy chicken suggesting its low pathogenicity nature. The virus has increased mammalian adaptation markers by mutation and intra- and inter-subtype reassortment, gathering gene segments possessing the most abundant maker mutations among previously circulating viruses. The increasing mammalian adaptation mutation in avian hosts suggests that they might be adaptive to infection in mammalian and avian hosts. It highlights the importance of genomic surveillance and adequate control measures for H5N1 infection in live poultry markets.

Risk assessment of the newly emerged H7N9 avian influenza viruses

ABSTRACT Since the first human case in 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1500 human infections with a mortality rate of approximately 40%. Despite large-scale poultry vaccination regimes across China, the H7N9 AIVs continue to persist and evolve rapidly in poultry. Recently, several strains of H7N9 AIVs have been isolated and shown the ability to escape vaccine-induced immunity. To assess the zoonotic risk of the recent H7N9 AIV isolates, we rescued viruses with hemagglutinin (HA) and neuraminidase (NA) from these H7N9 AIVs and six internal segments from PR8 virus and characterized their receptor binding, pH of fusion, thermal stability, plaque morphology and in ovo virus replication. We also assessed the cross-reactivity of the viruses with human monoclonal antibodies (mAbs) against H7N9 HA and ferret antisera against H7N9 AIV candidate vaccines. The H7N9 AIVs from the early epidemic waves had dual sialic acid receptor binding characteristics, whereas the more recent H7N9 AIVs completely lost or retained only weak human sialic acid receptor binding. Compared with the H7N9 AIVs from the first epidemic wave, the 2020/21 viruses formed larger plaques in Madin-Darby canine kidney (MDCK) cells and replicated to higher titres in ovo, demonstrating increased acid stability but reduced thermal stability. Further analysis showed that these recent H7N9 AIVs had poor cross-reactivity with the human mAbs and ferret antisera, highlighting the need to update the vaccine candidates. To conclude, the newly emerged H7N9 AIVs showed characteristics of typical AIVs, posing reduced zoonotic risk but a heightened threat for poultry.

A new PEDV strain CH/HLJJS/2022 can challenge current detection methods and vaccines

BackgroundPorcine epidemic diarrhea virus (PEDV) variant strains cause great economic losses to the global swine industry. However, vaccines do not provide sufficient protection against currently circulating strains due to viral mutations. This study traced the molecular characteristics of the most recent isolates in China and aimed to provide a basis for the prevention and treatment of PEDV.MethodsWe obtained samples from a Chinese diarrheal swine farm in 2022. Reverse transcription polymerase chain reaction and immunofluorescence were used to determine the etiology, and the full-length PEDV genome was sequenced. Nucleotide similarity was calculated using MEGA to construct a phylogenetic tree and DNASTAR. Mutant amino acids were aligned using DNAMAN and modeled by SWISS-MODEL, Phyre2 and FirstGlance in JMOL for protein tertiary structure simulation. Additionally, TMHMM was used for protein function prediction.ResultsA PEDV virulent strain CH/HLJJS/2022 was successfully isolated in China. A genome-wide based phylogenetic analysis suggests that it belongs to the GII subtype, and 96.1–98.9% homology existed in the whole genomes of other strains. For the first time, simultaneous mutations of four amino acids were found in the highly conserved membrane (M) and nucleocapsid (N) proteins, as well as eight amino acid mutations that differed from the vast majority of strains in the spike (S) protein. Three of the mutations alter the S-protein spatial structure. In addition, typing markers exist during strain evolution, but isolates are using the fusion of specific amino acids from multiple variant strains to add additional features, as also demonstrated by protein alignments and 3D models of numerous subtype strains.ConclusionThe newly isolated prevalent strain CH/HLJJS/2022 belonged to the GII subtype, and thirteen mutations different from other strains were found, including mutations in the highly conserved m and N proteins, and in the S1° and COE neutralizing epitopes of the S protein. PEDV is breaking through original cognitions and moving on a more complex path. Surveillance for PEDV now and in the future and improvements derived from mutant strain vaccines are highly warranted.

Genome Sequences of Three Common Bacterial Isolates from Wastewater from the Water Processor Assembly at the International Space Station.

Bacteria from the genera Burkholderia, Ralstonia, and Methylobacterium were consistently detected in water of the life support systems at the International Space Station. Here, we report complete genomes of recent isolates that are representative of these genera to support future studies in biofilm and wastewater treatment in space habitats.

1714. In Vitro Activity of Tebipenem Against Clinically Significant Gram-Positive Bacteria Isolated from Patients with Cancer

Abstract Background Carbapenems represent the most potent and broad-spectrum β-lactams and are minimally affected by most β-lactamase enzymes. however, all carbapenems currently in use including meropenem and ertapenem, are parenterally administered. Tebipenem pivoxil (TBPM-PI-HBr) is a new broad-spectrum orally-administered antibiotic, from the carbapenem subgroup of β-lactam antibiotics. It is the only oral carbapenem and could be an alternative to intravenous injection carbapenem antibiotic therapy. This study aimed to evaluate the in vitro activity of tebipenem, a novel oral carbapenem, against recent gram-positive clinical isolates from cancer patients. Methods We tested 300 gram-positive isolates are recently isolated (2019-2021) from our cancer patients, collected from blood cultures against tebipenem and comparators. Clinical and laboratory Standards Instituted (CLSI) approved broth microdilution method was used with appropriate ATCC controls. MIC50, MIC90, MIC ranges and percent of susceptibility calculations were made using FDA breakpoints when available. There is no tebipenem susceptibility breakpoint for gram-positive isolates currently. MIC90 and MICs range of tebipenem and comparators against 300 gram-positive isolates from cancer patients Results MIC90 and range of tebipenem and comparators are shown in table(1). Tebipenem gave the lowest MIC90 and MIC ranges against most gram-positive isolates including Bacillus species, Corynebacterium species, Enterococcus faecalis, Micrococcus species, MSSA S.aureus, Staphylococcus lugdunensis. β-hemolytic Streptococcus, Streptococcus pneumonie, Viridans group streptococci, and Rothia species. The comparative potency of tebipenem vs meropenem as a carbapenem in this study showed that, tebipenem MIC values were generally lower at least 2-4 fold. Conclusion Our data demonstrate that oral tebipenem has promising activity against clinically significant bacterial pathogens isolated from cancer patients and it has compatible activity to that of meropenem as a carbapenem in this study. Further clinical evolution for oral carbapenem treatment of bacterial infections is warranted. Disclosures All Authors: No reported disclosures.

1693. Meropenem-Xeruborbactam: In Vitro Potency against Gram-Negative Bacteria in Comparison with Marketed and Investigational Beta-lactam (BL)/Beta-lactamase Inhibitor (BLI) Combinations

Abstract Background Several BLI combinations have entered into clinical testing or been approved for use but there are few comparative studies of their in vitro activity. Xeruborbactam (XER, formerly QPX7728) is a cyclic boronate BLI with potent inhibitory activity against serine and metallo-beta-lactamases (MBL). It is in clinical development in combination with meropenem (MEM). The objective of this study was to compare in vitro potency of MEM-XER to that of other investigational and approved BL/BLI combinations in head-to-head testing against recent isolates of carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii (CRAB) as well as Pseudomonas aeruginosa (PA). Methods The test panel of recent worldwide isolates consisted of 507 CRE including 168 MBL producers (157 NDM, 11 VIM, 1 IMP), 506 CRAB, and 506 PA that were representative of current isolates and included 65 MDR (resistant to ≥ 3 antimicrobial classes) strains. MEM-XER was tested with XER at a fixed 8 mg/L conc. and other BL/BLI combinations at their recommended BLI concentrations. MIC testing was conducted blinded to drug identity using CLSI reference methods and controlled using approved quality control bacterial strains and ranges. Results The MIC50 and MIC90 for MEM-XER vs CRE ranged from ≤ 0.03 mg/L to 0.06 mg/L and 0.06 mg/L to 1 mg/L, respectively. MEM-XER and cefepime-taniborbactam (FEP-TANI) were the only BL/BLIs with activity against MBL-producers; based on MIC90, MEM-XER (MIC90 of 1 mg/L) was 16-fold more potent than FEP-TANI (MIC90 of 16 mg/L). MEM-XER and sulbactam-durlobactam (SUL-DUR) were the only agents with high potency against CRAB, with MIC50/MIC90 of 0.5/4 mg/L and 1/2 mg/L. For the representative panel of PA, the potency of MEM-XER, FEP-TAN, ceftolozane-tazobactam (TOL-TAZ), imipenem-relebactam (IMP-REL), and CAZ-AVI was similar with MIC90s ranging between 2-8 mg/L; for the MDR panel, MIC90‘s were > 8 mg/L for all combinations, with MER-XER, IMP-REL, TOL-TAZ, and CAZ-AVI having MIC50’s of 2-4 mg/L. Conclusion Compared to the marketed or investigational BL/BLIs, MEM-XERU had the broadest coverage of carbapenem-resistant gram-negative bacteria, with the most differentiating property being activity against multiple strains of MBL-producing CRE and CRAB. Disclosures Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Jill Lindley, BS, AbbVie: Grant/Research Support.

358. Surveillance of Activity of Antifungals: Data from the Analysis of Resistance in Antifungals (ARIA) 2020 Study

Abstract Background ARIA is a new annual global surveillance initiative collecting yeast and fungal isolates from worldwide designed to determine resistance to antifungal agents and trends over time. The ARIA program was developed in 2020 to provide a repository of recent clinical fungal isolates with known susceptibility profiles and to monitor resistance trends over time. ARIA reports the susceptibility patterns of its earliest data concerning echinocandins, second-generation triazoles, and fluconazole against clinical Candida albicans, non-albicans strains including C. auris, Aspergillus and Fusarium isolates from worldwide sources. Methods Isolates were collected from hospital worldwide during 2020, shipped to a central laboratory and re-identified by MALDI-TOF or molecular methods. MIC tests were performed by broth microdilution method in line with CLSI susceptibility testing standards. Percentage of susceptibility was calculated according to CLSI breakpoints. Antifungals tested were amphotericin B (AMB), anidulafungin (ANID), fluconazole (FLU), isavuconazole (ISA), micafungin (MIC), posaconazole (POS), and voriconazole (VOR). All testing was performed according to CLSI M27-A4 and M38-A2 methodologies. Results See table. Conclusion Ongoing antifungal resistance surveillance like the ARIA program is of utmost importance in order to monitor the efficacy of traditional empirical therapy and for the development of novel antifungal agents. This repository and ongoing ARIA program will provide a resource to better support the biopharmaceutical industry’s goals to develop new and more potent antifungal agents. Disclosures All Authors: No reported disclosures.

2176. In Vitro Evaluation of Delafloxacin Activity Against Contemporary US Isolates from Cystic Fibrosis Patients Hospitalized with Pneumonia: Results from the SENTRY Antimicrobial Surveillance Program (2019-2021)

Abstract Background Delafloxacin (DLX) is a broad-spectrum fluoroquinolone antibacterial approved in the US for the treatment of community-acquired bacterial pneumonia (CABP) and acute bacterial skin and skin structure infections. DLX is indicated to treat CABP caused by multiple pathogens, including methicillin-susceptible Staphylococcus aureus (MSSA) and Pseudomonas aeruginosa (PSA). S. aureus (SA) and PSA are common pathogens causing pneumonia in cystic fibrosis (CF) patients. In this study, the in vitro susceptibilities of DLX and comparator quinolones were determined for clinical isolates from US CF patients collected during 2019-2021. Methods Isolates from CF patients hospitalized with pneumonia were consecutively collected at 17 US medical centers participating in the SENTRY Surveillance Program. Sites submitted 1 isolate per patient per infection episode. Isolate identification was determined at each site and confirmed using MALDI-TOF at JMI Laboratories. Susceptibility testing was performed according to CLSI broth microdilution methodology. FDA interpretive criteria were used for DLX, and CLSI (2022) criteria were applied to comparators. Results A total of 115 SA, including 72 MSSA and 67 PSA, were submitted. Susceptibilities (%S) to DLX, levofloxacin (LEV), and moxifloxacin (MOX) for MSSA are shown in the table. As MOX does not have breakpoints for PSA, ciprofloxacin (CIP) was tested. Against all SA, %S was 73.9%, 67.0%, and 67.0% for DLX, LEV, and MOX, respectively. DLX had the highest %S against MSSA (94.4%). The %S to LEV and MOX was 87.5% and 87.5%. DLX was also more active than comparators against PSA, with DLX 68.7%S, while LEV was 43.3%S and CIP was 50.7%S. Conclusion DLX had good activity against recent CF isolates from US hospitals, and had the highest percent susceptibility of the quinolones tested against MSSA and PSA. These in vitro data suggest that DLX could be a useful therapy when coverage of both MSSA and PSA is needed. Disclosures Dee Shortridge, PhD, AbbVie: Grant/Research Support|JMI Laboratory: Employee|Melinta: Grant/Research Support|Menarini: Grant/Research Support|Shionogi: Grant/Research Support Jennifer M. Streit, BS, MT(ASCP), Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Michael D. Huband, BS, AbbVie: Grant/Research Support|Melinta: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support.

Identification and characterization of novel lineage 1 Powassan virus strains in New York State

ABSTRACT Powassan virus (POWV, family Flaviviridae) is a reemerging tick-borne virus endemic in North America and Russia. In 1997, a POWV-like agent was isolated from Ixodes scapularis in New England and determined to be genetically distinct from the original POWV isolate. This revealed the existence of two lineages: lineage 1, prototype Powassan virus (POWV-1) and lineage 2, deer tick virus (DTV). POWV-1 is thought to be primarily maintained in a cycle between I. cookei and woodchucks and I. marxi and squirrels, while DTV is primarily maintained in a cycle between I. scapularis and small mammal hosts. Recent tick, mammalian, and human isolates from New York State (NYS) have been identified as DTV, but for the first time in 45 years, we detected four POWV-1 isolates, including the first reported isolation of POWV-1 from I. scapularis. We aimed to investigate genotypic and phenotypic characteristics of recent NYS isolates through sequence analysis and evaluation of replication kinetics in vitro and in vivo. Our sequencing revealed genetic divergence between NYS POWV-1 isolates, with two distinct foci. We found that POWV-1 isolates displayed variable replication kinetics in nymphal ticks but not in cell culture. POWV-1 isolated from I. scapularis displayed increased fitness in experimentally infected I. scapularis as compared to historic and recent POWV-1 isolates from I. cookei. These data suggest the emergence of divergent POWV-1 strains in alternate tick hosts and maintenance of genetically and phenotypically discrete POWV-1 foci.

Efficacy of DNA Vaccines in Protecting Rainbow Trout against VHS and IHN under Intensive Farming Conditions.

Despite the negative impact of viral hemorrhagic septicemia (VHS) and infectious hematopoietic necrosis (IHN) on European rainbow trout farming, no vaccines are commercially available in Europe. DNA vaccines are protective under experimental conditions, but testing under intensive farming conditions remains uninvestigated. Two DNA vaccines encoding the glycoproteins (G) of recent Italian VHSV and IHNV isolates were developed and tested for potency and safety under experimental conditions. Subsequently, a field vaccination trial was initiated at a disease-free hatchery. The fish were injected intramuscularly with either the VHS DNA vaccine or with a mix of VHS and IHN DNA vaccines at a dose of 1 µg/vaccine/fish, or with PBS. At 60 days post-vaccination, fish were moved to a VHSV and IHNV infected facility. Mortality started 7 days later, initially due to VHS. After 3 months, IHN became the dominant cause of disease. Accordingly, both DNA vaccinated groups displayed lower losses compared to the PBS group during the first three months, while the VHS/IHN vaccinated group subsequently had the lowest mortality. A later outbreak of ERM caused equal disease in all groups. The trial confirmed the DNA vaccines to be safe and efficient in reducing the impact of VHS and IHN in farmed rainbow trout.

Plasmodium falciparum Sexual Commitment Rate Variation among Clinical Isolates and Diverse Laboratory-Adapted Lines.

Asexual blood-stage malaria parasites must produce sexual progeny to infect mosquitoes. It is important to understand the scope and causes of intraspecific variation in sexual commitment rates, particularly for the major human parasite P. falciparum. First, two alternative assay methods of measuring sexual commitment were compared to test a genetically modified P. falciparum line with elevated commitment rates inducible by overexpression of GDV1. The methods yielded correlated measurements with higher sensitivity and precision being achieved by one employing detection of the early gametocyte differentiation marker Pfs16. Thus, this was used to survey a diverse range of parasite lines and test each in multiple biological replicate assays in a serum-free medium supplemented with Albumax. There were differences among six recent clinical isolates from Ghana in their mean rates of sexual commitment per cycle, ranging from 3.3% to 12.2%. Among 13 diverse long-term laboratory-adapted lines, mean sexual commitment rates for most ranged from 4.7% to 13.4%, a few had lower rates with means from 0.3 to 1.6%, and one with a nonfunctional ap2-g gene always showed zero commitment. Among a subset of lines tested for the effects of exogenous choline to suppress commitment, there were significant differences. As expected, there was no effect in a line that had lost the gdv1 gene and that had generally low commitment, whereas the others showed quantitatively variable but significant responses to choline, suggesting potential trait variation. The results indicated the value of performing multiple replicate assays for understanding the variation of this key reproductive trait that likely affects transmission. IMPORTANCE Only sexual-stage malaria parasites are transmitted from human blood to mosquitoes. Thus, it is vital to understand variations in sexual commitment rates because these may be modifiable or susceptible to blocking. Two different methods of commitment rate measurement were first compared, demonstrating higher sensitivity and precision by the detection of an early differentiation marker, which was subsequently used to survey diverse lines. Clinical isolates from Ghana showed significant variation in mean per-cycle commitment rates and variation among biological replicates. Laboratory-adapted lines of diverse origins had a wider range with most being within the range observed for the clinical isolates, while a minority consistently had lower or zero rates. There was quantitative variation in the effects when adding choline to suppress commitment, indicating differing responsiveness of parasites to this environmental modification. Performing multiple assay replicates and comparisons of diverse isolates was important to understand this trait and its potential effects on transmission.

Macrolide Resistance in Bordetella pertussis: Current Situation and Future Challenges.

Pertussis is a highly contagious respiratory infection caused by Bordetella pertussis bacterium. The mainstay of treatment is macrolide antibiotics that reduce transmissibility, shorten the duration of symptoms and decrease mortality in infants. Recently, the macrolide resistance of B. pertussis has been reported globally but is especially widespread in mainland China. In this review, we aim to summarise the current understanding of the epidemiology, resistance mechanisms and clinical implications of B. pertussis macrolide resistance. Since the first appearance of macrolide-resistant B. pertussis in Arizona, USA, in 1994, only sporadic cases have been reported outside China. In certain parts of China, on the other hand, up to 70-100% of the recent clinical isolates have been found to be macrolide resistant. Reasons for macrolide resistance being centred upon China during the last decade can only be speculated on, but the dominant B. pertussis lineage is different between China and most of the high-income countries. It seems evident that efforts to increase awareness, guide molecular epidemiological surveillance and carry out systematic screening of B. pertussis positive samples for macrolide resistance should be implemented globally. In addition, practices to improve the clinical care of infants with pertussis caused by resistant strains should be studied vigorously.

Arboviruses as an unappreciated cause of non-malarial acute febrile illness in the Dschang Health District of western Cameroon.

Acute febrile illness is a common problem managed by clinicians and health systems globally, particularly in the Tropics. In many regions, malaria is a leading and potentially deadly cause of fever; however, myriad alternative etiologies exist. Identifying the cause of fever allows optimal management, but this depends on many factors including thorough knowledge of circulating infections. Arboviruses such as dengue (DENV) cause fever and may be underdiagnosed in sub-Saharan Africa where malaria is a major focus. We examined cases of fever in western Cameroon that tested negative for malaria and found 13.5% (13/96) were due to DENV, with 75% (9/12) of these being DENV serotype 2 infections. Two complete DENV2 genomes were obtained and clustered closely to recent isolates from Senegal and Burkina Faso. The seroprevalence of DENV in this region was 24.8% (96/387). Neutralizing antibodies to DENV2 were detected in all (15/15) seropositive samples tested. Chikungunya (CHIKV) is an arthritogenic alphavirus that is transmitted by Aedes mosquitoes, the same principal vector as DENV. The seroprevalence for CHIKV was 15.7% (67/427); however, CHIKV did not cause a single case of fever in the 96 subjects tested. Of note, being seropositive for one arbovirus was associated with being seropositive for the other (Χ2 = 16.8, p<0.001). Taken together, these data indicate that Aedes-transmitted arboviruses are endemic in western Cameroon and are likely a common but underappreciated cause of febrile illness. This work supports the need for additional study of arboviruses in sub-Saharan Africa and efforts to improve diagnostic capacity, surveillance systems, and arbovirus prevention strategies.