Targeted amplification and genetic sequencing of the severe acute respiratory syndrome coronavirus 2 surface glycoprotein.

IMPORTANCE: The COVID-19 pandemic was accompanied by an unprecedented surveillance effort. The resulting data were and will continue to be critical for surveillance and control of SARS-CoV-2. However, some genomic surveillance methods experienced challenges as the virus evolved, resulting in incomplete and poor quality data. Complete and quality coverage, especially of the S-gene, is important for supporting the selection of vaccine candidates. As such, we developed a robust method to target the S-gene for amplification and sequencing. By focusing on the S-gene and imposing strict coverage and quality metrics, we hope to increase the quality of surveillance data for this continually evolving gene. Our technique is currently being deployed globally to partner laboratories, and public health representatives from 79 countries have received hands-on training and support. Expanding access to quality surveillance methods will undoubtedly lead to earlier detection of novel variants and better inform vaccine strain selection.

Qualitative Conceptual Content Analysis of COVID-19 Vaccine Administration Error Inquiries.

The launch of the COVID-19 vaccination program was the largest vaccination campaign in U.S. history, with an unprecedented demand for vaccine and new vaccination providers, warranting significant education and communication efforts. NIP-INFO (nipinfo@cdc.gov) is the Centers for Disease Control and Prevention's (CDC's) immunization inquiry response service, and it receives inquiries for COVID-19 and routine non-COVID vaccines. A qualitative analysis of NIP-INFO's content was performed to better characterize and understand some of the knowledge gaps and reasons that COVID-19 vaccine administration errors occur. A total of 734 COVID-19 vaccine administration error inquiries were received between January 2021 and April 2022. The most frequent inquiries related to storage (n = 191; 26.0%), incorrect dosage or product (n = 190; 25.9%), unauthorized age group (n = 108; 14.7%), and schedule (n = 105; 14.3%). Training and communication strategies are imperative to ensure proper vaccine administration and build and maintain vaccine confidence.

Effectiveness of two and three mRNA COVID-19 vaccine doses against Omicron- and Delta-Related outpatient illness among adults, October 2021-February 2022.

Background: We estimated SARS-CoV-2 Delta- and Omicron-specific effectiveness of two and three mRNA COVID-19 vaccine doses in adults against symptomatic illness in US outpatient settings. Methods: Between October 1, 2021, and February 12, 2022, research staff consented and enrolled eligible participants who had fever, cough, or loss of taste or smell and sought outpatient medical care or clinical SARS-CoV-2 testing within 10 days of illness onset. Using the test-negative design, we compared the odds of receiving two or three mRNA COVID-19 vaccine doses among SARS-CoV-2 cases versus controls using logistic regression. Regression models were adjusted for study site, age, onset week, and prior SARS-CoV-2 infection. Vaccine effectiveness (VE) was calculated as (1 - adjusted odds ratio) × 100%. Results: Among 3847 participants included for analysis, 574 (32%) of 1775 tested positive for SARS-CoV-2 during the Delta predominant period and 1006 (56%) of 1794 participants tested positive during the Omicron predominant period. When Delta predominated, VE against symptomatic illness in outpatient settings was 63% (95% CI: 51% to 72%) among mRNA two-dose recipients and 96% (95% CI: 93% to 98%) for three-dose recipients. When Omicron predominated, VE was 21% (95% CI: -6% to 41%) among two-dose recipients and 62% (95% CI: 48% to 72%) among three-dose recipients. Conclusions: In this adult population, three mRNA COVID-19 vaccine doses provided substantial protection against symptomatic illness in outpatient settings when the Omicron variant became the predominant cause of COVID-19 in the United States. These findings support the recommendation for a third mRNA COVID-19 vaccine dose.

Tibial baseplate position and posterior cruciate ligament status impact patient-reported outcomes in conforming dual-pivot bearing total knee arthroplasty.

BACKGROUND: In an effort to optimize clinical outcomes and enhance stability, ultracongruent bearings have been increasingly used in primary total knee arthroplasty (TKA). The importance of the posterior cruciate ligament (PCL) and optimal sagittal tibial baseplate position in ultracongruent bearing TKA remains unknown. This study sought to determine whether these modifiable, surgical-technique-dependent variables meaningfully impact patient-reported outcome measures. METHODS: A total of 759 primary TKAs of the same dual-pivot design performed using a consistent surgical technique between January 2016 and April 2019 were retrospectively reviewed. PCL status was recorded, and anteroposterior (AP) tibial baseplate position and posterior tibial slope were measured by two independent blinded raters. Patient-reported outcomes related to pain, function, satisfaction, and activity level were analyzed in relationship to PCL status, posterior tibial slope, and AP tibial baseplate position, in addition to other pertinent covariates. RESULTS: Median age and body mass index of the cohort were 68.3 years and 33.4 kg/m2, respectively, with 73% being female. In multivariate analysis, partial or full release of the PCL was predictive of a knee "always" feeling normal (odds ratio 1.42, P = .041). Furthermore, tibial baseplate position closer to the middle of the tibia was associated with greater improvements in pain with level walking, pain while climbing stairs, and Knee Injury and Osteoarthritis Outcome Score for Joint Replacement total scores (P ≤ .079). CONCLUSION: In congruent dual-pivot bearing TKA, partially or fully releasing the PCL and AP tibial baseplate position closer to the middle of the tibia may provide greater improvement in pain and function scores at minimum 1-year follow-up.

Zika Virus Infection Induces DNA Damage Response in Human Neural Progenitors That Enhances Viral Replication.

Zika virus (ZIKV) infection attenuates the growth of human neural progenitor cells (hNPCs). As these hNPCs generate the cortical neurons during early brain development, the ZIKV-mediated growth retardation potentially contributes to the neurodevelopmental defects of the congenital Zika syndrome. Here, we investigate the mechanism by which ZIKV manipulates the cell cycle in hNPCs and the functional consequence of cell cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that ZIKV, but not dengue virus (DENV), induces DNA double-strand breaks (DSBs), triggering the DNA damage response through the ATM/Chk2 signaling pathway while suppressing the ATR/Chk1 signaling pathway. Furthermore, ZIKV infection impedes the progression of cells through S phase, thereby preventing the completion of host DNA replication. Recapitulation of the S-phase arrest state with inhibitors led to an increase in ZIKV replication, but not of West Nile virus or DENV. Our data identify ZIKV's ability to induce DSBs and suppress host DNA replication, which results in a cellular environment favorable for its replication.IMPORTANCE Clinically, Zika virus (ZIKV) infection can lead to developmental defects in the cortex of the fetal brain. How ZIKV triggers this event in developing neural cells is not well understood at a molecular level and likely requires many contributing factors. ZIKV efficiently infects human neural progenitor cells (hNPCs) and leads to growth arrest of these cells, which are critical for brain development. Here, we demonstrate that infection with ZIKV, but not dengue virus, disrupts the cell cycle of hNPCs by halting DNA replication during S phase and inducing DNA damage. We further show that ZIKV infection activates the ATM/Chk2 checkpoint but prevents the activation of another checkpoint, the ATR/Chk1 pathway. These results unravel an intriguing mechanism by which an RNA virus interrupts host DNA replication. Finally, by mimicking virus-induced S-phase arrest, we show that ZIKV manipulates the cell cycle to benefit viral replication.

Insertion position as well as the inserted TRS and gene sequences differentially affect the retention of foreign gene expression by simian hemorrhagic fever virus (SHFV).

Recombinant SHFV infectious cDNA clones expressing a foreign gene from an additional sg mRNA were constructed. Two 3' genomic region sites, between ORF4' and ORF2b and between ORF4 and ORF5, were utilized for insertion of the myxoma M013 gene with a C-terminal V5 tag followed by one of the three inserted transcription regulatory sequences (TRS), TRS2', TRS4' or TRS7. M013 insertion at the ORF4'/ORF2b site but not the ORF4/ORF5 site generated progeny virus but only the recombinant virus with an inserted TRS2' retained the entire M013 gene through passage four. Insertion of an auto-fluorescent protein gene, iLOV, with an inserted TRS2' at the ORF4'/ORF2b site, generated viable progeny virus. iLOV expression was maintained through passage eight. Although regulation of SHFV subgenomic RNA synthesis is complex, the ORF4'/ORF2b site, which is located between the two sets of minor structural proteins, is able to tolerate foreign gene insertion.

Expanded subgenomic mRNA transcriptome and coding capacity of a nidovirus.

Members of the order Nidovirales express their structural protein ORFs from a nested set of 3' subgenomic mRNAs (sg mRNAs), and for most of these ORFs, a single genomic transcription regulatory sequence (TRS) was identified. Nine TRSs were previously reported for the arterivirus Simian hemorrhagic fever virus (SHFV). In the present study, which was facilitated by next-generation sequencing, 96 SHFV body TRSs were identified that were functional in both infected MA104 cells and macaque macrophages. The abundance of sg mRNAs produced from individual TRSs was consistent over time in the two different cell types. Most of the TRSs are located in the genomic 3' region, but some are in the 5' ORF1a/1b region and provide alternative sources of nonstructural proteins. Multiple functional TRSs were identified for the majority of the SHFV 3' ORFs, and four previously identified TRSs were found not to be the predominant ones used. A third of the TRSs generated sg mRNAs with variant leader-body junction sequences. Sg mRNAs encoding E', GP2, or ORF5a as their 5' ORF as well as sg mRNAs encoding six previously unreported alternative frame ORFs or 14 previously unreported C-terminal ORFs of known proteins were also identified. Mutation of the start codon of two C-terminal ORFs in an infectious clone reduced virus yield. Mass spectrometry detected one previously unreported protein and suggested translation of some of the C-terminal ORFs. The results reveal the complexity of the transcriptional regulatory mechanism and expanded coding capacity for SHFV, which may also be characteristic of other nidoviruses.

Microbial Pathogens in Fresh Produce -- the Regulatory Perspective.

The potential for microbial contamination of fruits and vegetables is high because of the wide variety of conditions to which the produce is exposed during growth, harvest, and distribution. Heat treatment may also destroy the protective barriers (peels, husks, rinds) of fruits and vegetables, permitting the entry of microbial pathogens into the produce and providing them access to nutrients essential for their growth and proliferation. Proper refrigeration, storage, and shipping conditions as well as removal of soil from fresh produce by washing with chlorinated water are recommended to prevent contamination.

Growth of Salmonella enteritidis in Yolk of Shell Eggs from Normal and Seropositive Hens.

The growth of Salmonella enteritidis inoculated into the yolks of shell eggs from normal and seropositive hens was determined at various temperatures. All eggs were inoculated with approximately 1 colony-forming unit (CFU)/g of yolk. In eggs from normal hens, the organism multiplied with a generation time of 25 min, reaching a density of about 108 CFU/g in 12 h at 37°C. A generation time of 3.5 h was observed in eggs incubated at 15.5°C, a temperature frequently used for commercial storage of eggs. Cell density of >107 CFU/g was reached in 4 d at 15.5°C. No multiplication was observed in eggs incubated at 7°C for 94 d. When inoculated eggs from seropositive birds were incubated at 37°C, the organism multiplied with a generation time of 35 min, reaching a cell density of >106 CFU/g in 12 h. Raw egg white was detrimental to cells, reducing cell viability 50% in 4 h at 37°C. The limulus amoebocyte lysate test gave a positive reaction with whole liquid egg containing <103 CFU/g. A protocol is suggested for possible application of this test in epidemiological studies that screen grade A shell eggs for Salmonella contamination.

Campylobacter jejuni and Campylobacter coli Production of a Cytotonic Toxin Immunologically Similar to Cholera Toxin.

An enzyme-linked immunosorbent assay (ELISA) based on binding to cholera toxin (CT) antibody was used to screen cell-free supernatant fluids from 11 strains of Campylobacter jejuni and one strain of Campylobacter coli . Positive results for seven of the eight clinical isolates as well as for one animal and one food isolate suggested that these strains produced an extracellular factor immunologically similar to CT. An affinity column (packed with Sepharose 4B conjugated to purified anti-CT IgG via cyanogen bromide) was used to separate the extracellular factor from cell-free supernatant fluids. Both unconcentrated supernatant fluids and affinity-purified material caused rounding in a Y-1 mouse adrenal cell assay, suggesting that the factor was a cytotonic toxin. Rounding of Y-1 cells caused by cell-free supernatant fluids, affinity-purified toxin or CT was neutralized by preincubation with CT or Campylobacter cytotonic toxin (CCT) antiserum. CCT and CT showed a reaction of partial identity by gel immunodiffusion, using IgG from CT antiserum. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) of purified CCT produced one band at 70,000 daltons. Cell-free concentrates were positive in the rabbit skin permeability test and caused fluid accumulation in rabbit ileal loops. However, cell-free supernatant fluids and concentrates heated at 90°C for 15 min and tested by the suckling mouse assay produced no fluid accumulation in the intestines of mice.