Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the etiologic agent associated with coronavirus disease, which emerged in late 2019. In response, we developed a ...diagnostic panel consisting of 3 real-time reverse transcription PCR assays targeting the nucleocapsid gene and evaluated use of these assays for detecting SARS-CoV-2 infection. All assays demonstrated a linear dynamic range of 8 orders of magnitude and an analytical limit of detection of 5 copies/reaction of quantified RNA transcripts and 1 x 10
50% tissue culture infectious dose/mL of cell-cultured SARS-CoV-2. All assays performed comparably with nasopharyngeal and oropharyngeal secretions, serum, and fecal specimens spiked with cultured virus. We obtained no false-positive amplifications with other human coronaviruses or common respiratory pathogens. Results from all 3 assays were highly correlated during clinical specimen testing. On February 4, 2020, the Food and Drug Administration issued an Emergency Use Authorization to enable emergency use of this panel.
On 29 March 2013, the Chinese Center for Disease Control and Prevention confirmed the first reported case of human infection with an avian influenza A(H7N9) virus. The recent human infections with ...H7N9 virus, totalling over 130 cases with 39 fatalities to date, have been characterized by severe pulmonary disease and acute respiratory distress syndrome (ARDS). This is concerning because H7 viruses have typically been associated with ocular disease in humans, rather than severe respiratory disease. This recent outbreak underscores the need to better understand the pathogenesis and transmission of these viruses in mammals. Here we assess the ability of A/Anhui/1/2013 and A/Shanghai/1/2013 (H7N9) viruses, isolated from fatal human cases, to cause disease in mice and ferrets and to transmit to naive animals. Both H7N9 viruses replicated to higher titre in human airway epithelial cells and in the respiratory tract of ferrets compared to a seasonal H3N2 virus. Moreover, the H7N9 viruses showed greater infectivity and lethality in mice compared to genetically related H7N9 and H9N2 viruses. The H7N9 viruses were readily transmitted to naive ferrets through direct contact but, unlike the seasonal H3N2 virus, did not transmit readily by respiratory droplets. The lack of efficient respiratory droplet transmission was corroborated by low receptor-binding specificity for human-like α2,6-linked sialosides. Our results indicate that H7N9 viruses have the capacity for efficient replication in mammals and human airway cells and highlight the need for continued public health surveillance of this emerging virus.
The emergence and spread of Zika virus (ZIKV) presented a challenge to the diagnosis of ZIKV infections in areas with transmission of dengue (DENV) and chikungunya (CHIKV) viruses. To facilitate ...detection of ZIKV infections, and differentiate these infections from DENV and CHIKV, we developed the Trioplex real-time RT-PCR assay (Trioplex assay). Here, we describe the optimization of multiplex and singleplex formats of the assay for a variety of chemistries and instruments to facilitate global standardization and implementation. We evaluated the analytical performance of all Trioplex modalities for detection of these three pathogens in serum and whole blood, and for ZIKV in urine. The limit of detection for the three viruses and in different RNA-extraction modalities is near 10
genome copy equivalents per milliliter (GCE/mL). Simultaneous testing of more than one specimen type from each patient provides a 6.4% additional diagnostic sensitivity. Overall, the high sensitivity of the Trioplex assay demonstrates the utility of this assay ascertaining Zika cases.
Abstract A(H3N2) influenza viruses have circulated in humans since 1968, and antigenic drift of the hemagglutinin (HA) protein continues to be a driving force that allows the virus to escape the ...human immune response. Since the major antigenic sites of the HA overlap into the receptor binding site (RBS) of the molecule, the virus constantly struggles to effectively adapt to host immune responses, without compromising its functionality. Here, we have structurally assessed the evolution of the A(H3N2) virus HA RBS, using an established recombinant expression system. Glycan binding specificities of nineteen A(H3N2) influenza virus HAs, each a component of the seasonal influenza vaccine between 1968 and 2012, were analyzed. Results suggest that while its receptor-binding site has evolved from one that can bind a broad range of human receptor analogs to one with a more restricted binding profile for longer glycans, the virus continues to circulate and transmit efficiently among humans.
Human infections by avian influenza A(H7N9) virus entail substantial morbidity and mortality. Treatment of infected patients with the neuraminidase (NA) inhibitor oseltamivir was associated with ...emergence of viruses carrying NA substitutions. In the NA inhibition (NI) assay, R292K conferred highly reduced inhibition by oseltamivir, while E119V and I222K each caused reduced inhibition. To facilitate establishment of laboratory correlates of clinically relevant resistance, experiments were conducted in ferrets infected with virus carrying wild-type or variant NA genes recovered from the A/Taiwan/1/2013 isolate. Oseltamivir treatment (5 or 25 mg/kg of body weight/dose) was given 4 h postinfection, followed by twice-daily treatment for 5 days. Treatment of ferrets infected with wild-type virus resulted in a modest dose-dependent reduction (0.7 to 1.5 log10 50% tissue culture infectious dose TCID50) in nasal wash viral titers and inflammation response. Conversely, treatment failed to significantly inhibit the replication of R292K or E119V virus. A small reduction of viral titers was detected on day 5 in ferrets infected with the I222K virus. The propensity for oseltamivir resistance emergence was assessed in oseltamivir-treated animals infected with wild-type virus; emergence of R292K virus was detected in 3 of 6 ferrets within 5 to 7 days postinfection. Collectively, we demonstrate that R292K, E119V, and I222K reduced the inhibitory activity of oseltamivir, not only in the NI assay, but also in infected ferrets, judged particularly by viral loads in nasal washes, and may signal the need for alternative therapeutics. Thus, these clinical outcomes measured in the ferret model may correlate with clinically relevant oseltamivir resistance in humans.
This report provides more evidence for using the ferret model to assess the susceptibility of influenza A(H7N9) viruses to oseltamivir, the most prescribed anti-influenza virus drug. The information gained can be used to assist in the establishment of laboratory correlates of human disease and drug therapy. The rapid emergence of viruses with R292K in treated ferrets correlates well with the multiple reports on this NA variant in treated human patients. Our findings highlight the importance of the discovery and characterization of new antiviral drugs with different mechanisms of action and the use of combination treatment strategies against emerging viruses with pandemic potential, such as avian H7N9 virus, particularly against those carrying drug resistance markers.
We report characteristics of oseltamivir-resistant influenza A(H1N1)pdm09 viruses and patients infected with these viruses in the United States. During 2013-14, fifty-nine (1.2%) of 4,968 analyzed US ...influenza A(H1N1)pdm09 viruses had the H275Y oseltamivir resistance-conferring neuraminidase substitution. Our results emphasize the need for local surveillance for neuraminidase inhibitor susceptibility among circulating influenza viruses.
Background. Patients contracting influenza A(H7N9) infection often developed severe disease causing respiratory failure. Neuraminidase (NA) inhibitors (NAIs) are the primary option for treatment, but ...information on drug-resistance markers for influenza A(H7N9) is limited. Methods. Four NA variants of A/Taiwan/1/2013(H7N9) virus containing a single substitution (NA-E119V, NAI222K, NA-I222R, or NA-R292K) recovered from an oseltamivir-treated patient were tested for NAI susceptibility in vitro; their replicative fitness was evaluated in cell culture, mice, and ferrets. Results. NA-R292K led to highly reduced inhibition by oseltamivir and peramivir, while NA-E119V, NA-I222K, and NA-I222R caused reduced inhibition by oseltamivir. Mice infected with any virus showed severe clinical signs with high mortality rates. NA-I222K virus was the most virulent in mice, whereas virus lacking NA change (NAWT) and NA-R292K virus seemed the least virulent. Sequence analysis suggests that PB2-S714N increased virulence of NA-I222K virus in mice; NS1-K126R, alone or in combination with PB2-V227M, produced contrasting effects in NA-WT and NA-R292K viruses. In ferrets, all viruses replicated to high titers in the upper respiratory tract but produced only mild illness. NA-R292K virus, showed reduced replicative fitness in this animal model. Conclusions. Our data highlight challenges in assessment of the replicative fitness of H7N9 NA variants that emerged in NAI-treated patients.
In 2015 and 2016, Colombia had a widespread outbreak of Zika virus. Data from two national population-based surveillance systems for symptomatic Zika virus disease (ZVD) and birth defects provided ...complementary information on the effect of the Zika virus outbreak on pregnancies and infant outcomes.
We collected national surveillance data regarding cases of pregnant women with ZVD that were reported during the period from June 2015 through July 2016. The presence of Zika virus RNA was identified in a subgroup of these women on real-time reverse-transcriptase-polymerase-chain-reaction (rRT-PCR) assay. Brain or eye defects in infants and fetuses and other adverse pregnancy outcomes were identified among the women who had laboratory-confirmed ZVD and for whom data were available regarding pregnancy outcomes. We compared the nationwide prevalence of brain and eye defects during the outbreak with the prevalence both before and after the outbreak period.
Of 18,117 pregnant women with ZVD, the presence of Zika virus was confirmed in 5926 (33%) on rRT-PCR. Of the 5673 pregnancies with laboratory-confirmed ZVD for which outcomes had been reported, 93 infants or fetuses (2%) had brain or eye defects. The incidence of brain or eye defects was higher among pregnancies in which the mother had an onset of ZVD symptoms in the first trimester than in those with an onset during the second or third trimester (3% vs. 1%). A total of 172 of 5673 pregnancies (3%) resulted in pregnancy loss; after the exclusion of pregnancies affected by birth defects, 409 of 5426 (8%) resulted in preterm birth and 333 of 5426 (6%) in low birth weight. The prevalence of brain or eye defects during the outbreak was 13 per 10,000 live births, as compared with a prevalence of 8 per 10,000 live births before the outbreak and 11 per 10,000 live births after the outbreak.
In pregnant women with laboratory-confirmed ZVD, brain or eye defects in infants or fetuses were more common during the Zika virus outbreak than during the periods immediately before and after the outbreak. The frequency of such defects was increased among women with a symptom onset early in pregnancy. (Funded by the Colombian Instituto Nacional de Salud and the Centers for Disease Control and Prevention.).
Point-of-care antigen tests are an important tool for SARS-CoV-2 detection. Antigen tests are less sensitive than real-time reverse transcriptase PCR (rRT-PCR). Data on the performance of the ...BinaxNOW antigen test compared to rRT-PCR and viral culture by symptom and known exposure status, timing during disease, or exposure period and demographic variables are limited. During 3 to 17 November 2020, we collected paired upper respiratory swab specimens to test for SARS-CoV-2 by rRT-PCR and Abbott BinaxNOW antigen test at two community testing sites in Pima County, Arizona. We administered a questionnaire to capture symptoms, known exposure status, and previous SARS-CoV-2 test results. Specimens positive by either test were analyzed by viral culture. Previously we showed overall BinaxNOW sensitivity was 52.5%. Here, we showed BinaxNOW sensitivity increased to 65.7% among currently symptomatic individuals reporting a known exposure. BinaxNOW sensitivity was lower among participants with a known exposure and previously symptomatic (32.4%) or never symptomatic (47.1%) within 14 days of testing. Sensitivity was 71.1% in participants within a week of symptom onset. In participants with a known exposure, sensitivity was highest 8 to 10 days postexposure (75%). The positive predictive value for recovery of virus in cell culture was 56.7% for BinaxNOW-positive and 35.4% for rRT-PCR-positive specimens. Result reporting time was 2.5 h for BinaxNOW and 26 h for rRT-PCR. Point-of-care antigen tests have a shorter turnaround time than laboratory-based nucleic acid amplification tests, which allows for more rapid identification of infected individuals. Antigen test sensitivity limitations are important to consider when developing a testing program.
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