Monoclonal Antibodies to S and N SARS-CoV-2 Proteins as Probes to Assess Structural and Antigenic Properties of Coronaviruses.

Antibodies targeting the spike (S) and nucleocapsid (N) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential tools. In addition to important roles in the treatment and diagnosis of infection, the availability of high-quality specific antibodies for the S and N proteins is essential to facilitate basic research of virus replication and in the characterization of mutations responsible for variants of concern. We have developed panels of mouse and rabbit monoclonal antibodies (mAbs) to the SARS-CoV-2 spike receptor-binding domain (S-RBD) and N protein for functional and antigenic analyses. The mAbs to the S-RBD were tested for neutralization of native SARS-CoV-2, with several exhibiting neutralizing activity. The panels of mAbs to the N protein were assessed for cross-reactivity with the SARS-CoV and Middle East respiratory syndrome (MERS)-CoV N proteins and could be subdivided into sets that showed unique specificity for SARS-CoV-2 N protein, cross-reactivity between SARS-CoV-2 and SARS-CoV N proteins only, or cross-reactivity to all three coronavirus N proteins tested. Partial mapping of N-reactive mAbs were conducted using truncated fragments of the SARS-CoV-2 N protein and revealed near complete coverage of the N protein. Collectively, these sets of mouse and rabbit monoclonal antibodies can be used to examine structure/function studies for N proteins and to define the surface location of virus neutralizing epitopes on the RBD of the S protein.

Visualizing Rous Sarcoma Virus Genomic RNA Dimerization in the Nucleus, Cytoplasm, and at the Plasma Membrane.

Retroviruses are unique in that they package their RNA genomes as non-covalently linked dimers. Failure to dimerize their genomes results in decreased infectivity and reduced packaging of genomic RNA into virus particles. Two models of retrovirus genome dimerization have been characterized: in murine leukemia virus (MLV), genomic RNA dimerization occurs co-transcriptionally in the nucleus, resulting in the preferential formation of genome homodimers; whereas in human immunodeficiency virus (HIV-1), genomic RNA dimerization occurs in the cytoplasm and at the plasma membrane, with a random distribution of heterodimers and homodimers. Although in vitro studies have identified the genomic RNA sequences that facilitate dimerization in Rous sarcoma virus (RSV), in vivo characterization of the location and preferences of genome dimerization has not been performed. In this study, we utilized three single molecule RNA imaging approaches to visualize genome dimers of RSV in cultured quail fibroblasts. The formation of genomic RNA heterodimers within cells was dependent on the presence of the dimerization initiation site (DIS) sequence in the L3 stem. Subcellular localization analysis revealed that heterodimers were present the nucleus, cytoplasm, and at the plasma membrane, indicating that genome dimers can form in the nucleus. Furthermore, single virion analysis revealed that RSV preferentially packages genome homodimers into virus particles. Therefore, the mechanism of RSV genomic RNA dimer formation appears more similar to MLV than HIV-1.

Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity.

Retroviruses package their full-length, dimeric genomic RNA (gRNA) via specific interactions between the Gag polyprotein and a "Ψ" packaging signal located in the gRNA 5'-UTR. Rous sarcoma virus (RSV) gRNA has a contiguous, well-defined Ψ element, that directs the packaging of heterologous RNAs efficiently. The simplicity of RSV Ψ makes it an informative model to examine the mechanism of retroviral gRNA packaging, which is incompletely understood. Little is known about the structure of dimerization initiation sites or specific Gag interaction sites of RSV gRNA. Using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), we probed the secondary structure of the entire RSV 5'-leader RNA for the first time. We identified a putative bipartite dimerization initiation signal (DIS), and mutation of both sites was required to significantly reduce dimerization in vitro. These mutations failed to reduce viral replication, suggesting that in vitro dimerization results do not strictly correlate with in vivo infectivity, possibly due to additional RNA interactions that maintain the dimers in cells. UV crosslinking-coupled SHAPE (XL-SHAPE) was next used to determine Gag-induced RNA conformational changes, revealing G218 as a critical Gag contact site. Overall, our results suggest that disruption of either of the DIS sequences does not reduce virus replication and reveal specific sites of Gag-RNA interactions.

Visualizing Association of the Retroviral Gag Protein with Unspliced Viral RNA in the Nucleus.

Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc Natl Acad Sci U S A 99:3944-3949, 2002, https://doi.org/10.1073/pnas.062652199; R. Garbitt-Hirst, S. P. Kenney, and L. J. Parent, J Virol 83:6790-6797, 2009, https://doi.org/10.1128/JVI.00101-09). These results raise the intriguing possibility that the primary contact between Gag and gRNA might occur in the nucleus. To examine this possibility, we created a RSV proviral construct that includes 24 tandem repeats of MS2 RNA stem-loops, making it possible to track RSV viral RNA (vRNA) in live cells in which a fluorophore-conjugated MS2 coat protein is coexpressed. Using confocal microscopy, we observed that both wild-type Gag and a nuclear export mutant (Gag.L219A) colocalized with vRNA in the nucleus. In live-cell time-lapse images, the wild-type Gag protein trafficked together with vRNA as a single ribonucleoprotein (RNP) complex in the nucleoplasm near the nuclear periphery, appearing to traverse the nuclear envelope into the cytoplasm. Furthermore, biophysical imaging methods suggest that Gag and the unspliced vRNA physically interact in the nucleus. Taken together, these data suggest that RSV Gag binds unspliced vRNA to export it from the nucleus, possibly for packaging into virions as the viral genome.IMPORTANCE Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex.

Toll-like receptor 7 mediates early innate immune responses to malaria.

Innate immune recognition of malaria parasites is the critical first step in the development of the host response. At present, Toll-like receptor 9 (TLR9) is thought to play a central role in sensing malaria infection. However, we and others have observed that Tlr9(-/-) mice, in contrast to mice deficient in the downstream adaptor, Myeloid differentiation primary response gene 88 (MYD88), exhibit few deficiencies in immune function during early infection with the malaria parasite Plasmodium chabaudi, implying that another MYD88-dependent receptor also contributes to the antimalarial response. Here we use candidate-based screening to identify TLR7 as a key sensor of early P. chabaudi infection. We show that TLR7 mediates a rapid systemic response to infection through induction of cytokines such as type I interferons (IFN-I), interleukin 12, and gamma interferon. TLR7 is also required for induction of IFN-I by other species and strains of Plasmodium, including an etiological agent of human disease, P. falciparum, suggesting that malaria parasites harbor a common pathogen-associated molecular pattern (PAMP) recognized by TLR7. In contrast to the nonredundant requirement for TLR7 in early immune activation, sensing through both TLR7 and TLR9 was required for proinflammatory cytokine production and immune cell activation during the peak of parasitemia. Our findings indicate that TLR7 plays a central role in early immune activation during malaria infection, whereas TLR7 and TLR9 contribute combinatorially to immune responses as infection progresses.

Experimental cross-species infection of common marmosets by titi monkey adenovirus.

Adenoviruses are DNA viruses that infect a number of vertebrate hosts and are associated with both sporadic and epidemic disease in humans. We previously identified a novel adenovirus, titi monkey adenovirus (TMAdV), as the cause of a fulminant pneumonia outbreak in a colony of titi monkeys (Callicebus cupreus) at a national primate center in 2009. Serological evidence of infection by TMAdV was also found in a human researcher at the facility and household family member, raising concerns for potential cross-species transmission of the virus. Here we present experimental evidence of cross-species TMAdV infection in common marmosets (Callithrix jacchus). Nasal inoculation of a cell cultured-adapted TMAdV strain into three marmosets produced an acute, mild respiratory illness characterized by low-grade fever, reduced activity, anorexia, and sneezing. An increase in virus-specific neutralization antibody titers accompanied the development of clinical signs. Although serially collected nasal swabs were positive for TMAdV for at least 8 days, all 3 infected marmosets spontaneously recovered by day 12 post-inoculation, and persistence of the virus in tissues could not be established. Thus, the pathogenesis of experimental inoculation of TMAdV in common marmosets resembled the mild, self-limiting respiratory infection typically seen in immunocompetent human hosts rather than the rapidly progressive, fatal pneumonia observed in 19 of 23 titi monkeys during the prior 2009 outbreak. These findings further establish the potential for adenovirus cross-species transmission and provide the basis for development of a monkey model useful for assessing the zoonotic potential of adenoviruses.

A novel adenovirus species associated with an acute respiratory outbreak in a baboon colony and evidence of coincident human infection.

Adenoviruses (AdVs) are DNA viruses that infect many vertebrate hosts, including humans and nonhuman primates. Here we identify a novel AdV species, provisionally named "simian adenovirus C (SAdV-C)," associated with a 1997 outbreak of acute respiratory illness in captive baboons (4 of 9) at a primate research facility in Texas. None of the six AdVs recovered from baboons (BaAdVs) during the outbreak, including the two baboons who died from pneumonia, were typeable. Since clinical samples from the two fatal cases were not available, whole-genome sequencing of nasal isolates from one sick baboon and three asymptomatic baboons during the outbreak was performed. Three AdVs were members of species SAdV-C (BaAdV-2 and BaAdV-4 were genetically identical, and BaAdV-3), while one (BaAdV-1) was a member of the recently described SAdV-B species. BaAdV-3 was the only AdV among the 4 isolated from a sick baboon, and thus was deemed to be the cause of the outbreak. Significant divergence (<58% amino acid identity) was found in one of the fiber proteins of BaAdV-3 relative to BaAdV-2 and -4, suggesting that BaAdV-3 may be a rare SAdV-C recombinant. Neutralizing antibodies to the other 3 AdVs, but not BaAdV-3, were detected in healthy baboons from 1996 to 2003 and staff personnel from 1997. These results implicate a novel adenovirus species (SAdV-C) in an acute respiratory outbreak in a baboon colony and underscore the potential for cross-species transmission of AdVs between humans and nonhuman primates. IMPORTANCE Adenoviruses (AdVs) are DNA viruses that infect many animals, including humans and monkeys. In 1997, an outbreak of acute respiratory illness from AdVs occurred in a baboon colony in Texas. Here we use whole-genome sequencing and antibody testing to investigate new AdVs in baboons (BaAdVs) during the outbreak, one of which, BaAdV-3, came from a sick animal. By sequence analysis, BaAdV-3 may be a recombinant strain that arose from a related BaAdV found in baboons nearby in the colony (who were not sick) and yet another unknown AdV. We also found antibodies to these new BaAdVs in baboons and staff personnel at the facility. Taken together, our findings of a new AdV species as the cause of an acute respiratory outbreak in a baboon colony underscore the ongoing threat from emerging viruses that may carry the potential for cross-species transmission between monkeys and humans.

Cross-species transmission of a novel adenovirus associated with a fulminant pneumonia outbreak in a new world monkey colony.

Adenoviruses are DNA viruses that naturally infect many vertebrates, including humans and monkeys, and cause a wide range of clinical illnesses in humans. Infection from individual strains has conventionally been thought to be species-specific. Here we applied the Virochip, a pan-viral microarray, to identify a novel adenovirus (TMAdV, titi monkey adenovirus) as the cause of a deadly outbreak in a closed colony of New World monkeys (titi monkeys; Callicebus cupreus) at the California National Primate Research Center (CNPRC). Among 65 titi monkeys housed in a building, 23 (34%) developed upper respiratory symptoms that progressed to fulminant pneumonia and hepatitis, and 19 of 23 monkeys, or 83% of those infected, died or were humanely euthanized. Whole-genome sequencing of TMAdV revealed that this adenovirus is a new species and highly divergent, sharing <57% pairwise nucleotide identity with other adenoviruses. Cultivation of TMAdV was successful in a human A549 lung adenocarcinoma cell line, but not in primary or established monkey kidney cells. At the onset of the outbreak, the researcher in closest contact with the monkeys developed an acute respiratory illness, with symptoms persisting for 4 weeks, and had a convalescent serum sample seropositive for TMAdV. A clinically ill family member, despite having no contact with the CNPRC, also tested positive, and screening of a set of 81 random adult blood donors from the Western United States detected TMAdV-specific neutralizing antibodies in 2 individuals (2/81, or 2.5%). These findings raise the possibility of zoonotic infection by TMAdV and human-to-human transmission of the virus in the population. Given the unusually high case fatality rate from the outbreak (83%), it is unlikely that titi monkeys are the native host species for TMAdV, and the natural reservoir of the virus is still unknown. The discovery of TMAdV, a novel adenovirus with the capacity to infect both monkeys and humans, suggests that adenoviruses should be monitored closely as potential causes of cross-species outbreaks.

Using a pan-viral microarray assay (Virochip) to screen clinical samples for viral pathogens.

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing. The Virochip has also been used to identify novel viruses, including the SARS coronavirus, a novel rhinovirus clade, XMRV (a retrovirus linked to prostate cancer), avian bornavirus (the cause of a wasting disease in parrots), and a novel cardiovirus in children with respiratory and diarrheal illness. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

A metagenomic analysis of pandemic influenza A (2009 H1N1) infection in patients from North America.

Although metagenomics has been previously employed for pathogen discovery, its cost and complexity have prevented its use as a practical front-line diagnostic for unknown infectious diseases. Here we demonstrate the utility of two metagenomics-based strategies, a pan-viral microarray (Virochip) and deep sequencing, for the identification and characterization of 2009 pandemic H1N1 influenza A virus. Using nasopharyngeal swabs collected during the earliest stages of the pandemic in Mexico, Canada, and the United States (n = 17), the Virochip was able to detect a novel virus most closely related to swine influenza viruses without a priori information. Deep sequencing yielded reads corresponding to 2009 H1N1 influenza in each sample (percentage of aligned sequences corresponding to 2009 H1N1 ranging from 0.0011% to 10.9%), with up to 97% coverage of the influenza genome in one sample. Detection of 2009 H1N1 by deep sequencing was possible even at titers near the limits of detection for specific RT-PCR, and the percentage of sequence reads was linearly correlated with virus titer. Deep sequencing also provided insights into the upper respiratory microbiota and host gene expression in response to 2009 H1N1 infection. An unbiased analysis combining sequence data from all 17 outbreak samples revealed that 90% of the 2009 H1N1 genome could be assembled de novo without the use of any reference sequence, including assembly of several near full-length genomic segments. These results indicate that a streamlined metagenomics detection strategy can potentially replace the multiple conventional diagnostic tests required to investigate an outbreak of a novel pathogen, and provide a blueprint for comprehensive diagnosis of unexplained acute illnesses or outbreaks in clinical and public health settings.

Thoracic CT findings of novel influenza A (H1N1) infection in immunocompromised patients.

The goal of this study is to describe the spectrum of initial and follow-up CT findings of novel influenza A (H1N1) infection in a series of immunocompromised patients. Eight immunocompromised patients with documented novel influenza A (H1N1) had CT imaging at our institution between May 2009 and August 2009. A total of 20 CTs (initial and follow-up) were reviewed for the presence, severity, and distribution of the following: ground glass opacity, consolidation, interlobular septal thickening, mosaic perfusion, airway wall thickening, airway dilatation, nodules, cysts, pleural effusion, pericardial effusion, lymphadenopathy, and air trapping. The most common findings were airway thickening/dilatation, peribronchial ground glass opacity, centrilobular nodules, and tree-in-bud opacities. Peripheral consolidation involving the lower lobes was also a common pattern. Findings frequently involved all lobes and were closely associated with either large or small airways. Two patients presented with atypical CT findings including focal lobar consolidation and patchy lower lobe consolidation with soft tissue centrilobular nodules. Most survivors showed near complete resolution of findings within 35 days. CT scans in immunocompromised patients with novel influenza H1N1 commonly show a strong airway predominance of findings or peripheral areas of consolidation involving the lower lobes. A subset of patients with novel influenza A (H1N1) will show findings not typical of viral infection.