Efficient extraction of adventitious virus nucleic acid using commercially available methods.

An essential step in pharmaceutical product development is screening for contamination with adventitious agents, and there is desire to develop highly sensitive assays to detect adventitious viral nucleic acid. This study sought to examine the nucleic acid extraction efficiency of three viral candidates in relevant background matrices using four different extraction methods. Three model adventitious viruses, Minute virus of Mice, Porcine Circovirus, and Feline Leukemia Virus, were diluted within a variety of background matrices relevant to pharmaceutical production methods. Upon extraction, the nucleic acid was quantified using droplet digital PCR methods. Four nucleic acid extraction methods were assessed, including commercially available kits and manual extraction methods. Each method recovered nucleic acid post-extraction for each of the model viruses within the tested background matrices. The silica-column based method recovered a greater amount of viral nucleic acid, compared to the other methods tested. Similar trends were observed when model virus was diluted in bioreactor supernatant, which replicates industry testing conditions and provides details on which extraction methods might be used in Next Generation Sequencing and PCR methods for detecting contamination within pharmaceutical products.

Rapid production and free distribution of a synthetic RNA material to support SARS-CoV-2 molecular diagnostic testing.

In response to the COVID-19 pandemic, the National Institute of Standards and Technology released a synthetic RNA material for SARS-CoV-2 in June 2020. The goal was to rapidly produce a material to support molecular diagnostic testing applications. This material, referred to as Research Grade Test Material 10169, was shipped free of charge to laboratories across the globe to provide a non-hazardous material for assay development and assay calibration. The material consisted of two unique regions of the SARS-CoV-2 genome approximately 4 kb nucleotides in length. The concentration of each synthetic fragment was measured using RT-dPCR methods and confirmed to be compatible with RT-qPCR methods. In this report, the preparation, stability, and limitations of this material are described.

A history of adventitious agent contamination and the current methods to detect and remove them from pharmaceutical products.

Preventing adventitious agents from contaminating pharmaceutical products has been an important goal of regulatory agencies and industry for decades. Contamination of these products does not only erode consumer trust but also can have potentially serious health consequences. There are a wide variety of adventitious agents that can contaminate many different classifications of products, with each combination requiring different techniques for prevention or detection of adventitious agent contamination. This review seeks to give a brief overview of adventitious agents that have contaminated released pharmaceutical products, explain the different products that are at risk of contamination, then describe the methods commonly used for the prevention and detection of adventitious agent contamination.

Simultaneous Coinfection of Macaques with Zika and Dengue Viruses Does not Enhance Acute Plasma Viremia but Leads to Activation of Monocyte Subsets and Biphasic Release of Pro-inflammatory Cytokines.

The consequences of simultaneous infection with Zika (ZIKV) and Dengue (DENV) viruses are poorly understood. Here we show that rhesus macaques experimentally coinfected simultaneously with ZIKV and DENV-2 demonstrated ZIKV or DENV replication without an enhancement of either infection. Coinfection was accompanied by an increase in the proportions of CD14+CD16+ pro-inflammatory subsets of monocytes and release of pro-inflammatory cytokines in the plasma. Numerous cytokines such as I-TAC, Eotaxin, RANTES, MCP-1, IFNγ and MIG demonstrated a biphasic peak that coincided with the differences in kinetics of ZIKV and DENV replication suggesting that viral replication likely differentially modulated the release of these cytokines. Red blood cell indices significantly declined during acute infection suggesting transient anemia, and was accompanied by elevated levels of muscle, liver and renal injury markers. These findings have implications for understanding the pathogenesis of coinfection in ZIKV and DENV endemic regions, and is the 1st report of an experimental coinfection using the rhesus macaque model for ZIKV and DENV infections.

The effect of Zika virus infection in the ferret.

Although initial observations of infections with the Zika virus describe a mild illness, more recent reports show that infections by Zika result in neurotropism. In 2015, substantial congenital malformations were observed, with numerous infants born with microcephaly in Brazil. To study the underlying mechanism and effects of the disease, it is critical to find suitable animal models. Rodents lack an immune system parallel to humans and also have lissencephalic brains, which are likely to react differently to infections. As the smallest gyrencephalic mammal, ferrets may provide an important animal model to study the Zika virus, as their brains share many characteristics with humans. To evaluate the prospect of using ferrets to study Zika virus infection, we injected seven pregnant jills with the PR strain subcutaneously on gestational day 21, corresponding to the initiation of corticogenesis. These injections resulted in mixed effects. Two animals died of apparent infection, and all kits were resorbed in another animal that did not die. The other four animals remained pregnant until gestational day 40, when the kits were delivered by caesarian section. We evaluated the animals using CT, MRI, diffusion tensor imaging, and immunohistochemistry. The kits displayed a number of features compatible with an infection that impacted both the brain and skull. The outcomes, however, were variable and differed within and across litters, which ranged from the absence of observable abnormalities to prominent changes, suggesting differential vulnerability of kits to infection by the Zika virus or to subsequent mechanisms of neurodevelopmental disruption.

Zika convalescent macaques display delayed induction of anamnestic cross-neutralizing antibody responses after dengue infection.

Structural similarities between Zika (ZIKV) and dengue virus (DENV) leads to the induction of cross-reactive responses. We have previously demonstrated that ZIKV exposed macaques significantly enhance DENV viremia. Here we show that this enhancement of DENV infection occurred in the presence of high levels of DENV cross-reactive IgG1 subclass of binding antibodies (bAb) with low DENV neutralizing antibody (nAb) activity (<1:10). The DENV-2 nAb titres after ZIKV infection were, however, higher than those induced in DENV-2 only infected animals suggesting that ZIKV induced low titres of cross-nAb against DENV. Surprisingly, DENV-2 infection of animals previously infected with ZIKV was not accompanied by an anamnestic increase in cross-nAb titres till about 1 week after DENV-2 infection. This delay coincided with enhanced DENV-2 viremia indicating that high levels of cross-bAb in the absence of high nAb contributes to enhancement of DENV infection. Serum collected 8 weeks after DENV-2 infection had high levels of nAb and showed delayed antibody dependent enhancement (ADE) of infection (1:100 dilution) as compared with serum that was collected from ZIKV infected animals prior to DENV-2 infection (1:10 dilution). Examination of serum from macaques that were simultaneously infected with both ZIKV and DENV-2 showed high levels of nAb and delayed ADE responses raising the possibility that the low levels of cross-nAb induced by ZIKV infection could be overcome by co-immunization against ZIKV and DENV infection. Taken together, our results provide additional insights into the nature and kinetics of cross-reactive antibody responses and identify a critical correlate that could potentially prevent enhancement of DENV infection during ZIKV convalescence.

Human Serum With High Neutralizing Antibody Titers Against Both Zika and Dengue Virus Shows Delayed In Vitro Antibody-Dependent Enhancement of Dengue Virus Infection.

Zika virus infection in a dengue virus-naïve subject was associated with the induction of high levels of cross-reactive binding antibodies. These responses were, however, largely non-neutralizing and displayed a capacity to enhance dengue infection in vitro at significantly low dilution (1:10). In contrast, a subject who had high levels of neutralizing antibodies against both dengue and Zika viruses enhanced infection at a dilution of 1:10 000. These results suggest that high levels of dengue cross-neutralizing antibodies could potentially prevent the enhancement of dengue infection in Zika virus-convalescent individuals.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum.

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

Prior Exposure to Zika Virus Significantly Enhances Peak Dengue-2 Viremia in Rhesus Macaques.

Structural and functional homologies between the Zika and Dengue viruses' envelope proteins raise the possibility that cross-reactive antibodies induced following Zika virus infection might enhance subsequent Dengue infection. Using the rhesus macaque model we show that prior infection with Zika virus leads to a significant enhancement of Dengue-2 viremia that is accompanied by neutropenia, lympocytosis, hyperglycemia, and higher reticulocyte counts, along with the activation of pro-inflammatory monocyte subsets and release of inflammatory mediators. Zika virus infection induced detectable Dengue cross-reactive serum IgG responses that significantly amplified after Dengue-2 virus infection. Serum from Zika virus immune animals collected prior to Dengue-2 infection showed significant capacity for in vitro antibody dependent enhancement of Dengue-1, 2, 3 and 4 serotypes suggesting that pre-existing immunity to Zika virus could potentially enhance infection by heterologous Dengue serotypes. Our results provide first in vivo evidence that prior exposure to Zika virus infection can enhance Dengue infection, which has implications for understanding pathogenesis and the development of vaccines.

The broad-spectrum antiviral compound ST-669 restricts chlamydial inclusion development and bacterial growth and localizes to host cell lipid droplets within treated cells.

Novel broad-spectrum antimicrobials are a critical component of a strategy for combating antibiotic-resistant pathogens. In this study, we explored the activity of the broad-spectrum antiviral compound ST-669 for activity against different intracellular bacteria and began a characterization of its mechanism of antimicrobial action. ST-669 inhibits the growth of three different species of chlamydia and the intracellular bacterium Coxiella burnetii in Vero and HeLa cells but not in McCoy (murine) cells. The antichlamydial and anti-C. burnetii activity spectrum was consistent with those observed for tested viruses, suggesting a common mechanism of action. Cycloheximide treatment in the presence of ST-669 abrogated the inhibitory effect, demonstrating that eukaryotic protein synthesis is required for tested activity. Immunofluorescence microscopy demonstrated that different chlamydiae grow atypically in the presence of ST-669, in a manner that suggests the compound affects inclusion formation and organization. Microscopic analysis of cells treated with a fluorescent derivative of ST-669 demonstrated that the compound localized to host cell lipid droplets but not to other organelles or the host cytosol. These results demonstrate that ST-669 affects intracellular growth in a host-cell-dependent manner and interrupts proper development of chlamydial inclusions, possibly through a lipid droplet-dependent process.