First Step Of Viral Infection Research Articles

Interaction of species A rotavirus VP4 with the cellular proteins vimentin and actin related protein 2 discovered by a proximity interactome assay.

Rotavirus (RV) is an important zoonosis virus, which can cause severe diarrhea and extra-intestinal infection. To date, some proteins or carbohydrates have been shown to participate in the attachment or internalization of RV, including HGBAs, Hsc70, and integrins. This study attempted to indicate whether there were other proteins that would participate in the entry of RV; thus, the RV VP4-interacting proteins were identified by proximity labeling. After analysis and verification, it was found that VIM and ACTR2 could significantly promote the proliferation of RV in intestinal cells. Through further viral binding assays after knockdown, antibody blocking, and recombinant protein overexpression, it was revealed that both VIM and ACTR2 could promote RV replication.

Heparan sulfate is the attachment factor associated with channel catfish virus infection on host cells.

Channel catfish virus (CCV; family Alloherpesviridae) infects channel catfish, causing great harm to aquaculture fisheries and economic development. Attachment is the first step in viral infection and relies on the interaction of virions with components of the extracellular matrix (ECM). The present study aimed to explored the role of the main three ECM components in CCV attachment. Western blotting and quantitative real-time PCR analysis showed that neither collagen nor hyaluronic acid treatments had significant effects on CCV attachment. When exogenous heparin was used as a competitive inhibitor, the adhesion of heparin sodium salt to CCV was dose-dependent. When the concentration of heparin sodium salt was 10 mg/mL, the inhibitory effect on CCV infection of channel catfish ovary (CCO/BB) cells was more than 90%. Heparinase I could significantly prevent CCV attachment by digesting heparan sulfate on the cell surface, and both heparin sodium salt and heparinase I could dose-dependently reduce CCV titers, suggesting that heparin plays an important role in CCV attachment. In addition, the binding experiments between heparin-agarose beads and virions showed that CCV virions could specifically bind to heparin in a dose-dependent manner. The above results suggested that heparan sulfate might be an attachment factor involved in CCV infection of CCO/BB cells. These results increase our understand of the attachment mechanism of CCV and lay the foundation for further research on antiviral drugs.

Computational investigation of the increased virulence and pathogenesis of SARS-CoV-2 lineage B.1.1.7.

New variants of SARS-CoV-2 are being reported worldwide. The World Health Organization has reported Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529) as the variants of concern. There are speculations that the variants might evade the host immune responses induced by currently available vaccines and develop resistance to drugs under consideration. The first step of viral infection in COVID-19 occurs through the interaction of the spike protein's receptor-binding domain (RBD) with the peptidase domain of the human ACE-2 (hACE-2) receptor. This study aims to get a molecular-level understanding of the mechanism behind the increased infection rate in the alpha variant. We have computationally studied the spike protein interaction in both the wild-type and B.1.1.7 variant with the hACE-2 receptor using molecular dynamics and MM-GBSA based binding free energy calculations. The binding free energy difference shows that the mutant variant of the spike protein has increased binding affinity for the hACE-2 receptor (i.e. ΔG(N501Y,A570D) is in the range -7.2 to -7.6 kcal mol-1) and the results were validated using Density functional theory. We demonstrate that with the use of state-of-the-art computational approaches, we can, in advance, predict the virulent nature of variants of SARS-CoV-2 and alert the world healthcare system.

COVID -19: EFFICACY AND SAFETY PROFILE OF MAIN VACCINES APPROVED FOR EMERGENCY USE AUTHORIZATION IN 2021

Coronavirus -2 (SARS-CoV-2), is a new member of the human coronaviruses family. It is a single-strand positive-strand nucleic acid (ssRNA), It also has  spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. The (S) protein is responsible for recognising and binding to receptors on the surface of host cells and plays an important role in the first step of viral infection. Coronavirus-2 is identified in late 2019 as the causative agent of a new disease (COVID-19) outbreak in China associated with severe medical complications and even death in some cases. In March 2020, the World Health Organization (WHO) announced the novel disease outbreak as a pandemic. Current Coronavirus pandemic is the most challenging health emergency to humanity in this century, with globally reported (184 .324 .026) confirmed cases and ( 3. 992. 680) confirmed deaths ,as of 7th  July 2021, according to WHO.
 Preventive measures of social distancing face masks wearing, hand washing and lockdown have slowed the spread of COVID-19, but the ideal one is vaccination to protect individuals and create everlasting change and return to normalcy. Efficacy and safety of vaccines is very essential to gain  public trust.
 There is a great variation in people’s confidence in vaccines that relies on several factors, including awareness about vaccines, its side effects, safety and efficacy.
 The objective of this review is to provide concise sound knowledge concerning the   main approved vaccines for protection against COVID -19 emerged from various phases of vaccines clinical trials and medical practices, to enhance public trust and to tackle vaccine skepticism .
  This study also intended to educate about the vaccine’s side effects, that may prompt individuals to receive the vaccine after comparing the acceptable side effects with the disease’s severity and there by promote communities confidence in the safety of COVID-19 vaccines.
 This review examines efficacy and safety profile of some authorized vaccines such as Pfizer/BioNTech mRNA, (BNT162b2), Moderna (mRNA-1273) ,Johnson & Johnson (Janssen), Oxford-AstraZeneca ChAdOx1-S, CoronaVac vaccine and Gam-COVID-Vac  (Sputnik V).
 The review concluded that currently authorized COVID-19 vaccines are effective and prevent serious COVID-19 symptoms and related hospitalization and death.  They are safe with acceptable minor local and systemic side effects, reported  by some recipients’ of the vaccine and are similar to the known side effects encountered with previously known vaccines such as seasonal influenza vaccine, as expected outcomes of immune response to the vaccines. The  reported serious side effects were very rare.

Viruses Like Sugars: How to Assess Glycan Involvement in Viral Attachment.

The first step of viral infection requires interaction with the host cell. Before finding the specific receptor that triggers entry, the majority of viruses interact with the glycocalyx. Identifying the carbohydrates that are specifically recognized by different viruses is important both for assessing the cellular tropism and for identifying new antiviral targets. Advances in the tools available for studying glycan–protein interactions have made it possible to identify them more rapidly; however, it is important to recognize the limitations of these methods in order to draw relevant conclusions. Here, we review different techniques: genetic screening, glycan arrays, enzymatic and pharmacological approaches, and surface plasmon resonance. We then detail the glycan interactions of enterovirus D68 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlighting the aspects that need further clarification.

Identification of SARS-CoV-2 Receptor Binding Inhibitors by In Vitro Screening of Drug Libraries.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the high-throughput screening of two compound libraries (LOPAC®1280 and DiscoveryProbeTM). Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD. In light of these findings, the screening system described herein can be applied to other drug libraries to discover potent SARS-CoV-2 inhibitors.

Genetic determinants of receptor-binding preference and zoonotic potential of H9N2 avian influenza viruses.

Receptor recognition and binding is the first step of viral infection and a key determinant of host specificity. The inability of avian influenza viruses to effectively bind human-like sialylated receptors is a major impediment to their efficient transmission in humans and pandemic capacity. Influenza H9N2 viruses are endemic in poultry across Asia and parts of Africa where they occasionally infect humans and are therefore considered viruses with zoonotic potential. We previously described H9N2 viruses, including several isolated from human zoonotic cases, showing a preference for human-like receptors. Here we take a mutagenesis approach, making viruses with single or multiple substitutions in H9 haemagglutinin and test binding to avian and human receptor analogues using biolayer interferometry. We determine the genetic basis of preferences for alternative avian receptors and for human-like receptors, describing amino acid motifs at positions 190, 226 and 227 that play a major role in determining receptor specificity, and several other residues such as 159, 188, 193, 196, 198 and 225 that play a smaller role. Furthermore, we show changes at residues 135, 137, 147, 157, 158, 184, 188, and 192 can also modulate virus receptor avidity and that substitutions that increased or decreased the net positive charge around the haemagglutinin receptor-binding site show increases and decreases in avidity, respectively. The motifs we identify as increasing preference for the human-receptor will help guide future H9N2 surveillance efforts and facilitate our understanding of the emergence of influenza viruses with increased zoonotic potential.IMPORTANCE As of 2020, over 60 infections of humans by H9N2 influenza viruses have been recorded in countries where the virus is endemic. Avian-like cellular receptors are the primary target for these viruses. However, given that human infections have been detected on an almost monthly basis since 2015, there may be a capacity for H9N2 viruses to evolve and gain the ability to target human-like cellular receptors. Here we identify molecular signatures that can cause viruses to bind human-like receptors, and we identify the molecular basis for the distinctive preference for sulphated receptors displayed by the majority of recent H9N2 viruses. This work will help guide future surveillance by providing markers that signify the emergence of viruses with enhanced zoonotic potential as well as improving understanding of the basis of influenza virus receptor-binding.

Prediction of Virus-Receptor Interactions Based on Improving Similarities.

Viral infectious diseases have been seriously threatening human health. The receptor binding is the first step of viral infection. Predicting virus-receptor interactions will be helpful for the interaction mechanism of viruses and receptors, and further find some effective ways of preventing and treating viral infectious diseases so as to reduce the morbidity and mortality caused by viruses. Some computation algorithms have been proposed for identifying potential virus-receptor interactions. However, a common problem in those methods is the presence of noise in the similarity network. A new computational model (Network Enhancement and the Regularized Least Squares [NERLS]) is proposed to predict virus-receptor interactions based on improving similarities by Network Enhancement (NE). NERLS integrates the virus sequence similarity, the receptor sequence similarity and known virus-receptor interactions. We compute the virus sequence similarity and known virus-receptor interactions to construct the virus similarity network. The receptor similarity network is constructed by the Gaussian interaction profile kernel similarity and the receptor sequence similarity. To obtain the final virus similarity network and the final receptor similarity network, NE is, respectively, applied for reducing the noise of the virus similarity network and the receptor similarity network. Finally, NERLS employs the regularized least squares to predict interactions of viruses and receptors. The experiment results show that NERLS achieves the area under curve value of 0.893 and 0.921 in 10-fold cross-validation and leave-one-out cross-validation, respectively, which is consistently superior to four related methods [which include Initial interaction scores method via the neighbors and the Laplacian regularized Least Square (IILLS), Bi-random walk on a heterogeneous network (BRWH), Laplacian regularized least squares classifier (LapRLS), and Collaborative matrix factorization (CMF)]. Furthermore, a case study also demonstrates that NERLS effectively predicts potential virus-receptor interactions.

Evidence for Gastrointestinal Infection of SARS-CoV-2

See Covering the Cover synopsis on page 1515. See Covering the Cover synopsis on page 1515. Since the novel coronavirus (SARS-CoV-2) was identified in Wuhan, China, at the end of 2019, the virus has spread to 32 countries, infecting more than 80,000 people and causing more than 2600 deaths globally. The viral infection causes a series of respiratory illnesses, including severe respiratory syndrome, indicating that the virus most likely infects respiratory epithelial cells and spreads mainly via respiratory tract from human to human. However, viral target cells and organs have not been fully determined, impeding our understanding of the pathogenesis of the viral infection and viral transmission routes. According to a recent case report, SARS-CoV-2 RNA was detected in a stool specimen,1Holshue M.L. et al.N Engl J Med. 2020; 382: 929-936Crossref PubMed Scopus (3818) Google Scholar raising the question of viral gastrointestinal infection and a fecal-oral transmission route. It has been proven that SARS-CoV-2 uses angiotensin-converting enzyme (ACE) 2 as a viral receptor for entry process.2Zhou P. et al.Nature. 2020; 579: 270-273Crossref PubMed Scopus (12486) Google Scholar ACE2 messenger RNA is highly expressed and stabilized by the neutral amino acid transporter B0AT1 (SLC6A19) in gastrointestinal system,3Harmer D. et al.FEBS Lett. 2002; 532: 107-110Crossref PubMed Scopus (600) Google Scholar,4Yan R. et al.Science. 2020; 367: 1444-1448Crossref PubMed Scopus (3014) Google Scholar providing a prerequisite for SARS-CoV-2 infection. To further investigate the clinical significance of SARS-CoV-2 RNA in feces, we examined the viral RNA in feces from 71 patients with SARS-CoV-2 infection during their hospitalizations. The viral RNA and viral nucleocapsid protein were examined in gastrointestinal tissues from 1 of the patients. From February 1 to 14, 2020, clinical specimens, including serum, nasopharyngeal, and oropharyngeal swabs; urine; stool; and tissues from 73 hospitalized patients infected with SARS-CoV-2 were obtained in accordance with China Disease Control and Prevention guidelines and tested for SARS-CoV-2 RNA by using the Chinese Center for Disease Control and Prevention–standardized quantitative polymerase chain reaction assay.5Li Q. et al.N Engl J Med. 2020; 382: 1199-1207Crossref PubMed Scopus (9428) Google Scholar Clinical characteristics of the 73 patients are shown in Supplementary Table 1. The esophageal, gastric, duodenal, and rectal tissues were obtained from 1 of the patients by using endoscopy. The patient’s clinical information is described in the Supplementary Case Clinical Information and Supplementary Table 2. Histologic staining (H&E) as well as viral receptor ACE2 and viral nucleocapsid staining were performed as described in the Supplementary Methods. The images of fluorescent staining were obtained by using laser scanning confocal microscopy (LSM880, Carl Zeiss MicroImaging, Oberkochen, Germany) and are shown in Figure 1. This study was approved by the Ethics Committee of The Fifth Affiliated Hospital, Sun Yat-sen University, and all patients signed informed consent forms. From February 1 to 14, 2020, among all of the 73 hospitalized patients infected with SARS-CoV-2, 39 (53.42%), including 25 male and 14 female patients, tested positive for SARS-CoV-2 RNA in stool, as shown in Supplementary Table 1. The age of patients with positive results for SARS-CoV-2 RNA in stool ranged from 10 months to 78 years old. The duration time of positive stool results ranged from 1 to 12 days. Furthermore, 17 (23.29%) patients continued to have positive results in stool after showing negative results in respiratory samples. Gastrointestinal endoscopy was performed on a patient as described in the Supplementary Case Clinical Information. As shown in Figure 1, the mucous epithelium of esophagus, stomach, duodenum, and rectum showed no significant damage with H&E staining. Infiltrate of occasional lymphocytes was observed in esophageal squamous epithelium. In lamina propria of the stomach, duodenum, and rectum, numerous infiltrating plasma cells and lymphocytes with interstitial edema were seen. Importantly, viral host receptor ACE2 stained positive mainly in the cytoplasm of gastrointestinal epithelial cells (Figure 1). We observed that ACE2 is rarely expressed in esophageal epithelium but is abundantly distributed in the cilia of the glandular epithelia. Staining of viral nucleocapsid protein was visualized in the cytoplasm of gastric, duodenal, and rectum glandular epithelial cell, but not in esophageal epithelium. The positive staining of ACE2 and SARS-CoV-2 was also observed in gastrointestinal epithelium from other patients who tested positive for SARS-CoV-2 RNA in feces (data not shown). In this article, we provide evidence for gastrointestinal infection of SARS-CoV-2 and its possible fecal-oral transmission route. Because viruses spread from infected to uninfected cells,6Xiao F. et al.PLoS Pathog. 2014; 10e1004128Crossref PubMed Scopus (79) Google Scholar viral-specific target cells or organs are determinants of viral transmission routes. Receptor-mediated viral entry into a host cell is the first step of viral infection. Our immunofluorescent data showed that ACE2 protein, which has been proven to be a cell receptor for SARS-CoV-2, is abundantly expressed in the glandular cells of gastric, duodenal, and rectal epithelia, supporting the entry of SARS-CoV-2 into the host cells. ACE2 staining is rarely seen in esophageal mucosa, probably because the esophageal epithelium is mainly composed of squamous epithelial cells, which express less ACE2 than glandular epithelial cells. Our results of SARS-CoV-2 RNA detection and intracellular staining of viral nucleocapsid protein in gastric, duodenal, and rectal epithelia demonstrate that SARS-CoV-2 infects these gastrointestinal glandular epithelial cells. Although viral RNA was also detected in esophageal mucous tissue, absence of viral nucleocapsid protein staining in esophageal mucosa indicates low viral infection in esophageal mucosa. After viral entry, virus-specific RNA and proteins are synthesized in the cytoplasm to assemble new virions,7Weiss S.R. et al.Microbiol Mol Biol Rev. 2005; 69: 635-664Crossref PubMed Scopus (769) Google Scholar which can be released to the gastrointestinal tract. The continuous positive detection of viral RNA from feces suggests that the infectious virions are secreted from the virus-infected gastrointestinal cells. Recently, we and others have isolated infectious SARS-CoV-2 from stool (unpublished data, 2020), confirming the release of the infectious virions to the gastrointestinal tract. Therefore, fecal-oral transmission could be an additional route for viral spread. Prevention of fecal-oral transmission should be taken into consideration to control the spread of the virus. Our results highlight the clinical significance of testing viral RNA in feces by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) because infectious virions released from the gastrointestinal tract can be monitored by the test. According to the current Centers for Disease Control and Prevention guidance for the disposition of patients with SARS-CoV-2, the decision to discontinue transmission-based precautions for hospitalized patients with SARS-CoV-2 is based on negative results rRT-PCR testing for SARS-CoV-2 from at least 2 sequential respiratory tract specimens collected ≥24 hours apart.8Centers for Disease Control and Preventionhttps://www.cdc.gov/coronavirus/2019-ncov/hcp/disposition-hospitalized-patients.htmlGoogle Scholar However, in more than 20% of patients with SARS-CoV-2, we observed that the test result for viral RNA remained positive in feces, even after test results for viral RNA in the respiratory tract converted to negative, indicating that the viral gastrointestinal infection and potential fecal-oral transmission can last even after viral clearance in the respiratory tract. Therefore, we strongly recommend that rRT-PCR testing for SARS-CoV-2 from feces should be performed routinely in patients with SARS-CoV-2 and that transmission-based precautions for hospitalized patients with SARS-CoV-2 should continue if feces test results are positive by rRT-PCR testing. On January 17, 2020, a 78-year-old man, who, along with his wife, had come from Wuhan 6 days earlier to visit his daughter, presented to the outpatient clinic at our hospital in Zhuhai, Guangdong Province, China, with a 7-day cough and fever. He was admitted to the negative-pressure isolation room in the Department of Infectious Diseases at our hospital as a suspected case of SARS-CoV-2 infection. On admission, the physical examination showed a body temperature of 37.5°C, blood pressure of 105/56 mm Hg, pulse of 67 beats per minute, and respiratory rate of 22 breaths per minute with oxygen saturation of 97%. On physical examination, auscultation indicated rhonchi and cracks on bilateral lungs. Initial arterial blood gas analysis showed the arterial partial pressure of oxygen (Pao2)/fraction of inspiration oxygen (Fio2) was 176 mmHg. Nasopharyngeal and oropharyngeal swab specimens tested positive by rRT-PCR for SARS-CoV-2. Chest computed tomography presented with multiple ground-glass opacities, coinciding with previous reports,1Kanne J.P. Chest CT Findings in 2019 Novel Coronavirus (2019-nCoV) Infections from Wuhan, China: Key Points for the Radiologist.Radiology. 2020; : 200241Google Scholar,2Huang C. Wang Y. Li X. et al.Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.Lancet. 2020; Abstract Full Text Full Text PDF Scopus (28324) Google Scholar showing evidence of pneumonia in both the left and right lungs. The patient’s wife and daughter tested positive for SARS-CoV-2 RNA and were admitted to the hospital on January 18, 2020. On hospital days 1 through 3, the patient remained febrile, with stable vital signs. The oxygen saturation remained above 95% with high-flow oxygen therapy. Empiric antimicrobials with oseltamivir and moxifloxacin was given during this period of time. On hospital day 4, the patient developed severe respiratory distress, with the Pao2/Fio2 decreasing to 130 mmHg and was immediately transferred to the intensive care unit, receiving an intubation and mechanical ventilation. Along with sedation, prone-position mechanical ventilation was applied for 12 hours per day, and low tidal volume was set. The Pao2/Fio2 increased to 350 mmHg immediately after intubation but decreased gradually again in the following several days to the lowest level of 70 at 10 days after admission. Meanwhile, the chest radiograph showed extensive bilateral consolidation; emergent veno-venous extracorporeal membrane oxygenation was applied at the same day. On day 10, coffee ground gastric contents were observed from the gastric drainage tube and fecal occult blood tested positive, indicating upper gastrointestinal bleed. Gastrointestinal endoscopy was performed to determine the exact location of bleeding. Mucosa damage in the esophagus was observed under endoscopy. Biopsy samples were taken from esophagus, gastric, duodenum, and colon for histopathologic and immunofluorescent staining. One day after treatment with octreotide, esomeprazole, etc, gastrointestinal bleeding stopped. As of February 12, 2020, the patient remained hospitalized. The vital signs were stable with mechanical ventilation, veno-venous extracorporeal membrane oxygenation, and low-dose vasopressors. There is no obvious evidence of other organ dysfunction. Esophageal, gastric, duodenal, and rectal tissues were obtained using endoscopy on day 10. Samples were embedded with paraffin and then stained with H&E. For immunofluorescent staining, 3-μm-thick sections were dewaxed in xylene, rehydrated in alcohol, and washed in distilled water 3 times before microwave repair. After washing 3 times in phosphate-buffered saline with Tween (PBST), sections were incubated with 10% goat serum in PBST for 1 hour at room temperature and then incubated overnight at 4°C with primary antibodies (anti-ACE2, Sino Biological, Beijing, China, 10108-T56, 1:500; anti-nucleoprotein, Sino Biological, 40143-T62, 1:500). The slides were incubated with secondary antibodies (Alexa Fluor 647–conjugated goat anti-rabbit IgG, bs-0296G-AF647, 1:100; Bioss, London, UK) for 1 hour at room temperature followed by washing 3 times with PBST. Nuclei were then counterstained with 4′,6-diamidino-2-phenylindole after washing 3 times with PBST. Slides were imaged by using a laser scanning confocal microscopy (LSM880, Carl Zeiss MicroImaging). Supplementary Table 1Clinical Characteristics of the 73 Hospitalized Patients Infected With SARS-CoV-2S+R+S+(R+S+/S+)%∼R+S+(∼R+S+/R+S+)%∼R-S+(∼R-S+/S+)%∼R-S-(∼R-S-/R+S+)%No. or % of patients733953.42%615.38%1743.59%1641.03%Female321443.75%214.29%535.71%750.00%Male412569.98%416.00%1248.00%936.00%Age (years)43 (0.83-7)49 (0.83-78)/52.5 (3-78)/44 (0.83-69)/47 (19-75)/Tumors7342.86%133.00%133.00%133.00%Surgical history17847.06%112.50%450.00%337.50%Ulcer00/0/0/0/Smoking9444%00250.00%250.00%Respiratory symptoms533056.60%413.33%1343.33%1343.33%Typical chest CT663654.55%513.89%1644.44%1541.67%Diarrhea261765.38%211.76%635.29%952.94%Gastrointestinal bleeding10440%125.00%125.00%250.00%Use of corticosteroid211257.14%216.67%325.00%758.33%Antibiotic therapy603552.05%617.14%1440.00%1542.86%Antiviral therapy733849.32%615.79%1642.11%1642.11%PPIs therapy512447.06%416.67%625.00%1458.33%NSAID12650.00%116.67%233.33%350.00%ICU44100%125.00%125.00%250.00%CT, computerized tomography; ICU, intensive care unit; NSAID, nonsteroidal anti-inflammatory drugs; PPI, proton pump inhibitor; R, respiratory specimens; R+, SARS-CoV-2 RNA tested positive in R on hospital admission; R+S+, SARS-CoV-2 RNA tested positive in both R and S during hospitalization; ∼R+S+, SARS-CoV-2 RNA remained positive in both R and S until the date of writing the manuscript on February 14th, 2020; ∼R-S+, SARS-CoV-2 RNA converted to negative in R during hospitalization but remained positive in S until the date of writing the manuscript on February 14th, 2020; ∼R-S-, SARS-CoV-2 RNA converted to negative in both R and S during hospitalization; S, stool specimens; S+, SARS-CoV-2 RNA tested positive in stool during hospitalization; /, not applicable. Open table in a new tab Supplementary Table 2Timeline of Detection of Viral RNA in Different Specimens of the Patient Infected With SARS-CoV-2SpecimenDay 1Day 2Day 3Day 5Day 7Day 9Day 10Day 11Day 13Day 14Day 16Day 18Day 20Day 21Day 22Day 24Day 26RespiratoryNTPositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositiveStoolNTNTNegativeNegativeNegativePositivePositivePositivePositivePositivePositiveNTPositivePositivePositivePositivePositiveSerumNTNTNegativeNegativeNegativeNegativeNegativePositiveNegativeNegativeNegativeNTNTNTNegativeNTNegativeUrineNTNTNegativeNegativeNegativeNegativeNegativeNTNegativeNTPositiveNTNTNTNTNTNTEsophagusNTNTNTNTNTNTPositiveNTNTNTNTNTNTNTNTNTNTStomachNTNTNTNTNTNTPositiveNTNTNTNTNTNTNTNTNTNTDuodenumNTNTNTNTNTNTPositiveNTNTNTNTNTNTNTNTNTNTRectumNTNTNTNTNTNTPositiveNTNTNTNTNTNTNTNTNTNTNT, denotes not tested. Open table in a new tab CT, computerized tomography; ICU, intensive care unit; NSAID, nonsteroidal anti-inflammatory drugs; PPI, proton pump inhibitor; R, respiratory specimens; R+, SARS-CoV-2 RNA tested positive in R on hospital admission; R+S+, SARS-CoV-2 RNA tested positive in both R and S during hospitalization; ∼R+S+, SARS-CoV-2 RNA remained positive in both R and S until the date of writing the manuscript on February 14th, 2020; ∼R-S+, SARS-CoV-2 RNA converted to negative in R during hospitalization but remained positive in S until the date of writing the manuscript on February 14th, 2020; ∼R-S-, SARS-CoV-2 RNA converted to negative in both R and S during hospitalization; S, stool specimens; S+, SARS-CoV-2 RNA tested positive in stool during hospitalization; /, not applicable. NT, denotes not tested. Covering the CoverGastroenterologyVol. 158Issue 6PreviewA Swedish nationwide study showed an increased risk of inflammatory bowel disease among individuals with microscopic colitis. Full-Text PDF Presence of SARS-Coronavirus-2 in the Ileal Mucosa: Another Evidence for Infection of GI Tract by This VirusGastroenterologyVol. 159Issue 4PreviewWe have read with great interest the recent article published entitled “Evidence for Gastrointestinal Infection of SARS-CoV-2.” In this article, Xiao et al1 report how ≤20% of patients with negative viral RNA in their respiratory tract present positive RNA in feces. They present a case of a patient with severe pneumonia owing to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and gastrointestinal bleeding, with viral RNA in the gastrointestinal mucosa, as well as the presence by immunofluorescence of abundant angiotensin-converting enzyme (ACE)2 receptors in the gastrointestinal tract. Full-Text PDF Fecal–Oral Transmission of SARS-COV-2: Practical ImplicationsGastroenterologyVol. 159Issue 4PreviewWe read with great interest the study by Xiao et al1 on evidence for gastrointestinal infection of coronavirus disease-19 (COVID-19). Testing for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA in stool specimens of 73 hospitalized patients resulted in virus detection in 53.4% of patients, both with and without gastrointestinal manifestations (ie, diarrhea, nausea, vomiting, gastrointestinal bleeding). In addition, COVID-19 nucleic acid was positive in feces of 23.3% of patients in which respiratory samples had already turned negative. Full-Text PDF Gastrointestinal ACE2, COVID-19 and IBD: Opportunity in the Face of Tragedy?GastroenterologyVol. 159Issue 4PreviewWe read with interest the articles by Xiao et al1 and Du et al2 regarding severe acute respiratory syndrome coronavirus-2 (SARS Co-V 2) shedding in feces, staining of viral nucleocapsid protein in the cytoplasm of gastrointestinal epithelial cells, and the characterization of angiotensin-converting enzyme 2 (ACE2) receptors across tissues in the human body. The relationship between coronavirus disease 2019 (COVID-19), intestinal ACE2 expression, and gastrointestinal symptoms is worth exploring further, and may offer unique clues to the pathogenesis of intestinal inflammation. Full-Text PDF

Structural Transition and Antibody Binding of EBOV GP and ZIKV E Proteins from Pre-Fusion to Fusion-Initiation State.

Membrane fusion proteins are responsible for viral entry into host cells—a crucial first step in viral infection. These proteins undergo large conformational changes from pre-fusion to fusion-initiation structures, and, despite differences in viral genomes and disease etiology, many fusion proteins are arranged as trimers. Structural information for both pre-fusion and fusion-initiation states is critical for understanding virus neutralization by the host immune system. In the case of Ebola virus glycoprotein (EBOV GP) and Zika virus envelope protein (ZIKV E), pre-fusion state structures have been identified experimentally, but only partial structures of fusion-initiation states have been described. While the fusion-initiation structure is in an energetically unfavorable state that is difficult to solve experimentally, the existing structural information combined with computational approaches enabled the modeling of fusion-initiation state structures of both proteins. These structural models provide an improved understanding of four different neutralizing antibodies in the prevention of viral host entry.

Grass carp reovirus-GD108 fiber protein is involved in cell attachment.

Viral attachment to specific host receptors is the first step in viral infection and serves an essential function in the selection of target cells. In this study, structure analysis, neutralization assays, and cell attachment assays were carried out to evaluate the cell attachment functions of the outer capsid fiber protein of grass carp reovirus GD108 strain (GCRV-GD108). The GCRV-GD108 fiber protein contained 512 amino acids encoded by S7 segment and shared sequence similarities with mammalian reovirus cell attachment protein σ1 and adenovirus fiber. Structural analyses predicted the presence of a coiled-coil tail domain, three adenoviral shafts in the body domain, and a globular head domain, similar to other fiber proteins. Neutralization assays showed that polyclonal antibodies against the fiber protein could prevent viral infection in both fish and grass carp snout fibroblast cells (PSF), suggesting that the recombinant fiber protein could induce neutralized antibodies against GCRV-GD108. Cell attachment assays showed that recombinant fiber protein could bind to PSF cells, demonstrating that the fiber protein functioned as the cell attachment protein in GCRV-GD108. These results provided the basis for further studies of the pathogenesis of grass carp reovirus.

Preliminary crystallographic analysis of the Megavirus superoxide dismutase.

Megavirus chilensis, a close relative of the Mimivirus giant virus, is able to replicate in Acanthamoeba castellanii. The first step of viral infection involves the internalization of the virions in host vacuoles. It has been experimentally demonstrated that Mimivirus particles contain many proteins capable of resisting oxidative stress, as encountered in the phagocytic process. These proteins are conserved in Megavirus, which has an additional gene (Mg277) encoding a putative superoxide dismutase. The Mg277 ORF product was overexpressed in Escherichia coli, purified and crystallized. A SAD data set was collected to 2.24 Å resolution at the selenium peak wavelength on the BM30 beamline at the ESRF from a single crystal of selenomethionine-substituted recombinant superoxide dismutase protein.

Mechanisms of Host Receptor Adaptation by Severe Acute Respiratory Syndrome Coronavirus

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.

Inhibition of hepatitis C virus infection by anti-claudin-1 antibodies is mediated by neutralization of E2-CD81-Claudin-1 associations

The tight junction protein claudin-1 (CLDN1) has been shown to be essential for hepatitis C virus (HCV) entry-the first step of viral infection. Due to the lack of neutralizing anti-CLDN1 antibodies, the role of CLDN1 in the viral entry process is poorly understood. In this study, we produced antibodies directed against the human CLDN1 extracellular loops by genetic immunization and used these antibodies to investigate the mechanistic role of CLDN1 for HCV entry in an infectious HCV cell culture system and human hepatocytes. Antibodies specific for cell surface-expressed CLDN1 specifically inhibit HCV infection in a dose-dependent manner. Antibodies specific for CLDN1, scavenger receptor B1, and CD81 show an additive neutralizing capacity compared with either agent used alone. Kinetic studies with anti-CLDN1 and anti-CD81 antibodies demonstrate that HCV interactions with both entry factors occur at a similar time in the internalization process. Anti-CLDN1 antibodies inhibit the binding of envelope glycoprotein E2 to HCV permissive cell lines in the absence of detectable CLDN1-E2 interaction. Using fluorescent-labeled entry factors and fluorescence resonance energy transfer methodology, we demonstrate that anti-CLDN1 antibodies inhibit CD81-CLDN1 association. In contrast, CLDN1-CLDN1 and CD81-CD81 associations were not modulated. Taken together, our results demonstrate that antibodies targeting CLDN1 neutralize HCV infectivity by reducing E2 association with the cell surface and disrupting CD81-CLDN1 interactions. These results further define the function of CLDN1 in the HCV entry process and highlight new antiviral strategies targeting E2-CD81-CLDN1 interactions.

Structure of Reovirus σ1 in Complex with Its Receptor Junctional Adhesion Molecule-A

Viral attachment to specific host receptors is the first step in viral infection and serves an essential function in the selection of target cells. Mammalian reoviruses are highly useful experimental models for studies of viral pathogenesis and show promise as vectors for oncolytics and vaccines. Reoviruses engage cells by binding to carbohydrates and the immunoglobulin superfamily member, junctional adhesion molecule-A (JAM-A). JAM-A exists at the cell surface as a homodimer formed by extensive contacts between its N-terminal immunoglobulin-like domains. We report the crystal structure of reovirus attachment protein σ1 in complex with a soluble form of JAM-A. The σ1 protein disrupts the JAM-A dimer, engaging a single JAM-A molecule via virtually the same interface that is used for JAM-A homodimerization. Thus, reovirus takes advantage of the adhesive nature of an immunoglobulin-superfamily receptor by usurping the ligand-binding site of this molecule to attach to the cell surface. The dissociation constant (KD) of the interaction between σ1 and JAM-A is 1,000-fold lower than that of the homophilic interaction between JAM-A molecules, indicating that JAM-A strongly prefers σ1 as a ligand. Analysis of reovirus mutants engineered by plasmid-based reverse genetics revealed residues in σ1 required for binding to JAM-A and infectivity of cultured cells. These studies define biophysical mechanisms of reovirus cell attachment and provide a platform for manipulating reovirus tropism to enhance vector targeting.

Squid axoplasm supports the retrograde axonal transport of herpes simplex virus.

Neurotropic viruses, such as Herpes simplex virus type 1 (HSV1), first enter the axon terminal and are transported to the cell body where they replicate in the nucleus. Based on previous studies using whole animal assays (1), as well as assays of whole cells in vitro (2, 3), we know that HSV enters axons and travels as an unenveloped particle–capsid plus associated tegument proteins–in a retrograde direction toward the cell body. The rate of transport has been estimated to be 3–5 mm/hr (2). Although these earlier studies provided important insight into the delivery of virus to the nucleus, they were limited. What has been needed is an experimental system in which HSV can be applied to a particular region of a neuron at a known concentration and time, and the transport of the virus assayed quantitatively within the axon. With such an assay one could address questions about the real time motility of the viral particle and about the viral and cellular proteins that are essential for this behavior. The giant axon of the squid, Loligo pealei, serves as a powerful model for the molecular mechanisms of axoplasmic transport. The microtubule-based motor, kinesin, was discovered in squid (4); and microtubule-based transport in both anterograde and retrograde directions has been extensively characterized. Recently, actin-based transport of squid axoplasmic organelles has also been described (5, 6). Organelles move in either direction in the giant axon at 2–4 μm/s. Organelles isolated from squid axons move towards the barbed ends of actin filaments (6, 7) at 1.6 μm/s (8) and in either direction on microtubules at 2 μm/s (9). The axon contains long tracks of microtubule-actin filament bundles that appear to serve collectively as tracks for axonal transport (10). Thus, the squid axon contains all the requisite molecular machinery for transport. In this report, we describe the use of the squid giant axon as an alternative to previous approaches and present for the first time direct observation of the movement of HSV in living axons. To image the virus in the axon, we used a viral HSV strain in which a major tegument protein, viral protein (VP) 16, was generated as a fusion protein with GFP at the C terminal. This virus was grown in Vero cells, purified, and concentrated to a titer of about 1.0 × 109 pfu/ml. Different aliquots of the viral stock were treated in various ways to remove the viral envelope. This mimics the first step in viral infection in which the viral envelope fuses with the cell membrane, releasing the capsid together with its tegument into the cytosol. In vitro removal of the envelope was necessary for these experiments because injection of virus into the axon by-passes the normal membrane fusion step. Several treatments were found that produced motile, fluorescent particles in the axon after injection. The viral preparation was co-injected with non-fluorescent oil. The oil droplet was used to determine an appropriate focal plane and as a fixed reference marker, since it remains stationary after injection (11). The movement of viral particles was recorded on a laser scanning confocal microscope. Movement was sampled ~1 frame/3 s, and transport rates were determined by analyzing sequential frames. When we examined fields upstream from the oil droplet, i.e., closer to the cell body, we identified rapidly moving particles. One of these moved ~36 μm in 18 s for an overall rate of 1.9 μm/s in the retrograde direction (Fig. 1). At the end of this sequence, this particle went out of the plane of focus. Of 113 moving particles examined, all were found in the region of the axon between the oil droplet and the cell body, moving in the retrograde direction. Figure 1 Retrograde transport of a GFP-labeled viral particle in a living squid axon. The giant axon was dissected in Ca++-containing seawater and injected with >100pfu of GFP-VP16 labeled and extracted Herpes simplex virus. Still frames were taken from ... The effect on the virus of the pre-injection treatment was determined by electron microscopy of negatively stained preparations. Of the virus in those samples that produced motile particles when injected in the axon, ~90% had lost their surface envelope. Since the GFP-labeled protein, VP16, is a tegument protein, the moving particles we observed must include at least tegument, either as whole virus, tegument plus capsid, or simply as tegument aggregates. Since neither capsid nor membrane are GFP-labeled, we cannot know from these studies whether they also are capable of moving. We have demonstrated the rapid and preferential retrograde axonal transport of HSV particles in the living axon. The rate of movement of these viral particles in the squid giant axon is similar to the estimated rates of retrograde transport of this human virus in rat dorsal root ganglia in culture (2). Furthermore, this rate is consistent with the rates of retrograde transport of endogenous organelles in the squid axon and of isolated organelles on either microtubules or actin filaments. Both the direction and rate of movement suggest that a host cell motor molecule, such as the retrograde microtubule motor, dynein, may be co-opted by invading virus. Thus, by combining the power of the squid axon with a biochemical and genetic dissection of the virus, we expect to be able to identify the viral proteins required for transport, as well as the cellular transport machinery that they recruit.

Sequence-specific inhibition of human immunodeficiency virus (HIV) reverse transcription by antisense oligonucleotides: comparative study in cell-free assays and in HIV-infected cells.

We have investigated two regions of the viral RNA of human immunodeficiency virus type 1 (HIV-1) as potential targets for antisense oligonucleotides. An oligodeoxynucleotide targeted to the U5 region of the viral genome was shown to block the elongation of cDNA synthesized by HIV-1 reverse transcriptase in vitro. This arrest of reverse transcription was independent of the presence of RNase H activity associated with the reverse transcriptase enzyme. A second oligodeoxynucleotide targeted to a site adjacent to the primer binding site inhibited reverse transcription in an RNase H-dependent manner. These two oligonucleotides were covalently linked to a poly(L-lysine) carrier and tested for their ability to inhibit HIV-1 infection in cell cultures. Both oligonucleotides inhibited virus production in a sequence- and dose-dependent manner. PCR analysis showed that they inhibited proviral DNA synthesis in infected cells. In contrast, an antisense oligonucleotide targeted to the tat sequence did not inhibit proviral DNA synthesis but inhibited viral production at a later step of virus development. These experiments show that antisense oligonucleotides targeted to two regions of HIV-1 viral RNA can inhibit the first step of viral infection--i.e., reverse transcription--and prevent the synthesis of proviral DNA in cell cultures.

Anti-viral activity of human recombinant heparin-binding proteins HBNF and MK

Herpes simplex viruses bind to cell surface heparan sulfate proteoglycans, as a first step of viral infection. We report here that two recombinant heparin-binding proteins HBNF and MK inhibit infectivity of human herpes simplex viruses types 1 and 2 and human cytomegalovirus. Carboxymethylated HBNF and MK, which retain affinity for heparin-Sepharose, do not exhibit anti-viral activities. Arguments are presented that anti-viral effects of HBNF and MK are due to the competition for the specific binding to the cell surface heparan sulfate proteoglycans.

Biochemical Characterization of a Glycoprotein Required for Rhinovirus Attachment

Human rhinoviruses attach to specific receptors located on the surfaces of host cells as a first step in viral infection. A 90-kDa cell surface protein was previously shown to be involved in the attachment of human rhinoviruses to susceptible cells (Tomassini, J. E., and Colonno, R.J. (1986) J. Virol. 58, 290-295). Digestion of purified receptor protein with various glycosidases revealed that 30% of its molecular mass was comprised of complex-type oligosaccharides, one-third being contributed by sialic acid. The presence of sialic acid was confirmed by demonstrating that wheat germ lectin can inhibit the attachment of rhinoviruses to host cell membranes, while lectins of other sugar specificities had no effect. The oligosaccharides were shown to be N-linked by tunicamycin treatment of host cells and by N-glycanase digestion. Seven N-linked glycosylation sites were detected by partial digestion of the receptor oligosaccharides with N-glycanase. Native receptor protein had an isoelectric focusing point of 4.2, compared to 5.3 for the deglycosylated protein. Studies of virus and antibody binding to neuraminidase-treated host cell membranes suggested that although carbohydrates may be involved in host-virus interaction, the receptor carbohydrate is not the predominant component of the cellular receptor site.