Coronavirus Infection-Associated Cell Death Signaling and Potential Therapeutic Targets

COVID-19 is the name of the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that occurred in 2019. The virus–host-specific interactions, molecular targets on host cell deaths, and the involved signaling are crucial issues, which become potential targets for treatment. Spike protein, angiotensin-converting enzyme 2 (ACE2), cathepsin L-cysteine peptidase, transmembrane protease serine 2 (TMPRSS2), nonstructural protein 1 (Nsp1), open reading frame 7a (ORF7a), viral main protease (3C-like protease (3CLpro) or Mpro), RNA dependent RNA polymerase (RdRp) (Nsp12), non-structural protein 13 (Nsp13) helicase, and papain-like proteinase (PLpro) are molecules associated with SARS-CoV infection and propagation. SARS-CoV-2 can induce host cell death via five kinds of regulated cell death, i.e., apoptosis, necroptosis, pyroptosis, autophagy, and PANoptosis. The mechanisms of these cell deaths are well established and can be disrupted by synthetic small molecules or natural products. There are a variety of compounds proven to play roles in the cell death inhibition, such as pan-caspase inhibitor (z-VAD-fmk) for apoptosis, necrostatin-1 for necroptosis, MCC950, a potent and specific inhibitor of the NLRP3 inflammasome in pyroptosis, and chloroquine/hydroxychloroquine, which can mitigate the corresponding cell death pathways. However, NF-κB signaling is another critical anti-apoptotic or survival route mediated by SARS-CoV-2. Such signaling promotes viral survival, proliferation, and inflammation by inducing the expression of apoptosis inhibitors such as Bcl-2 and XIAP, as well as cytokines, e.g., TNF. As a result, tiny natural compounds functioning as proteasome inhibitors such as celastrol and curcumin can be used to modify NF-κB signaling, providing a responsible method for treating SARS-CoV-2-infected patients. The natural constituents that aid in inhibiting viral infection, progression, and amplification of coronaviruses are also emphasized, which are in the groups of alkaloids, flavonoids, terpenoids, diarylheptanoids, and anthraquinones. Natural constituents derived from medicinal herbs have anti-inflammatory and antiviral properties, as well as inhibitory effects, on the viral life cycle, including viral entry, replication, assembly, and release of COVID-19 virions. The phytochemicals contain a high potential for COVID-19 treatment. As a result, SARS-CoV-2-infected cell death processes and signaling might be of high efficacy for therapeutic targeting effects and yielding encouraging outcomes.

Case Report: Dengue Hemorrhagic Fever with Ischemic Stroke

Several neurological manifestations are recognized in dengue infection, but stroke is a rare complication. We report a case of ischemic stroke in a patient with dengue hemorrhagic fever. A 52-year-old previously healthy male presented with a history of fever for 2 days, and left-sided weakness and numbness of sudden onset. MRI scanning showed a right-sided thalamic lacunar infarct. Diagnosis of dengue fever was made based on leuco-thrombocytopenia, positive dengue nonstructural protein-1 (NS-1) antigen, and positive dengue IgM antibodies. Severity of limb weakness correlated with the critical phase of dengue hemorrhagic fever (DHF). He was discharged home with good recovery from neurological symptoms and disability. Strokes are rare in dengue, and are mainly hemorrhagic strokes related to thrombocytopenia. Ischemic stroke is even rarer. More evidence is needed for confirmation of dengue as a pathogenic mechanism of ischemic stroke.

Clinically observed deletions in SARS-CoV-2 Nsp1 affect protein stability and its ability to inhibit translation

Nonstructural protein 1 (Nsp1) is a major pathogenicity factor of SARS-CoV-2. It inhibits host-cell translation, primarily through a direct interaction between its C-terminal domain and the mRNA entry channel of the 40S small ribosomal subunit, with an N-terminal β-barrel domain fine-tuning the inhibition and promoting selective translation of viral mRNA. SARS-CoV-2 nsp1 is a target of recurring deletions, some of which are associated with altered COVID-19 disease progression. To provide the biochemical basis for this, it is essential to characterize the efficiency of translational inhibition by the said protein variants. Here, we use an in vitro translation system to investigate the translation inhibition capacity of a series of clinically observed Nsp1 deletion variants. We find that a frequently observed deletion of residues 79-89 destabilized the N-terminal domain (NTD) and severely reduced the capacity of Nsp1 to inhibit translation. Interestingly, shorter deletions in the same region have been reported to effect the type I interferon response but did not affect translation inhibition, indicating a possible translation-independent role of the Nsp1 NTD in interferon response modulation. Taken together, our data provide a mechanistic basis for understanding how deletions in Nsp1 influence SARS-CoV-2 induction of interferon response and COVID-19 progression.

Structural analysis of SARS-Cov-2 nonstructural protein 1 polymorphisms found in the Brazilian Amazon

The coronavirus disease COVID-19 has been the cause of millions of deaths worldwide. Among the SARS-CoV-2 proteins, the non-structural protein 1 (NSP1) has great importance during the virus infection process and is present in both alpha and beta-CoVs. Therefore, monitoring of NSP1 polymorphisms is crucial in order to understand their role during infection and virus-induced pathogenicity. Herein, we analyzed how mutations detected in the circulating SARS-CoV-2 in the population of the city of Manaus, Amazonas state, Brazil could modify the tertiary structure of the NSP1 protein. Three mutations were detected in the SARS-CoV-2 NSP1 gene: deletion of the amino acids KSF from positions 141 to 143 (delKSF), SARS-CoV-2, lineage B.1.195; and two substitutions, R29H and R43C, SARS-CoV-2 lineage B.1.1.28 and B.1.1.33, respectively. The delKSF was found in 47 samples, whereas R29H and R43C were found in two samples, one for each mutation. The NSP1 structures carrying the mutations R43C and R29H on the N-terminal portion (e.g. residues 10 to 127) showed minor backbone divergence compared to the Wuhan model. However, the NSP1 C-terminal region (residues 145 to 180) was severely affected in the delKSF and R29H mutants. The intermediate variable region (residues 144 to 148) leads to changes in the C-terminal region, particularly in the delKSF structure. New investigations must be carried out to analyze how these changes affect NSP1 activity during the infection. Our results reinforce the need for continuous genomic surveillance of SARS-CoV-2 to better understand virus evolution and assess the potential impact of the viral mutations on the approved vaccines and future therapies.

DENGUE FEVER OUTBREAK IN TWIN CITIES; A TERTIARY CARE CENTER EXPERIENCE

Objective: To characterize the clinical presentations and laboratory markers (as disease predictors and markers) and severity of disease in patients admitted with dengue fever. Study Design: Prospective observational study. Place and Duration of Study: Department of Infectious Diseases, Pak Emirates Military Hospital Rawalpindi, from May to Oct 2018 Methodology: In our study, we included the patients treated for dengue fever. Data were recorded on predesigned proforma and analyzed using Microsoft excel 365 and Social Package for Statistical Sciences version 23. Results: A total of 160 patients were included in this study, all were males. All cases were positive for Nonstructural Protein 1 Antigen. Symptoms seen in abundance were fever with rigors, chills, backache, retro-orbital pain, nausea and vomiting. Manual platelet count at admission ranged from 15-396 x 103 (mean 107.5 ± 6.8 x 103). Platelet count on discharge ranged from 102-577 x 103 (mean 207.9 ± 95.4 x 103). A total of 34 (21.25%) patients out of 160 confirmed and admitted dengue fever cases had Dengue Hemorrhagic Fever (Nonstructural Protein 1 Antigen positive, thrombocytopenia and leukopenia) and 2 patient went into dengue shock syndrome. The derangement in alanine aminotransferase and alkaline phosphatase levels were shown in 98 (61.2%) and 62 (31.2%) of patients. All these patients were immunoglobulin M and Nonstructural Protein 1Antigen positive. Conclusion: Raised serum alanine aminotransferase and alkaline phosphatase levels along with early positive Nonstructural Protein 1 Antigen is a marker of disease severity.

Genotypic Variants of Pandemic H1N1 Influenza A Viruses Isolated from Severe Acute Respiratory Infections in Ukraine during the 2015/16 Influenza Season

Human type A influenza viruses A(H1N1)pdm09 have caused seasonal epidemics of influenza since the 2009–2010 pandemic. A(H1N1)pdm09 viruses had a leading role in the severe epidemic season of 2015/16 in the Northern Hemisphere and caused a high incidence of acute respiratory infection (ARI) in Ukraine. Serious complications of influenza-associated severe ARI (SARI) were observed in the very young and individuals at increased risk, and 391 fatal cases occurred in the 2015/16 epidemic season. We analyzed the genetic changes in the genomes of A(H1N1)pdm09 influenza viruses isolated from SARI cases in Ukraine during the 2015/16 season. The viral hemagglutinin (HA) fell in H1 group 6B.1 for all but four isolates, with known mutations affecting glycosylation, the Sa antigenic site (S162N in all 6B.1 isolates), or virulence (D222G/N in two isolates). Other mutations occurred in antigenic site Ca (A141P and S236P), and a subgroup of four strains were in group 6B.2, with potential alterations to antigenicity in A(H1N1)pdm09 viruses circulating in 2015/16 in Ukraine. A cluster of Ukrainian isolates exhibited novel D2E and N48S mutations in the RNA binding domain, and E125D in the effector domain, of immune evasion nonstructural protein 1 (NS1). The diverse spectrum of amino-acid substitutions in HA, NS1, and other viral proteins including nucleoprotein (NP) and the polymerase complex suggested the concurrent circulation of multiple lineages of A(H1N1)pdm09 influenza viruses in the human population in Ukraine, a country with low vaccination coverage, complicating public health measures against influenza.

Antiviral Activity of Canine RIG-I against Canine Influenza Virus and Interactions between Canine RIG-I and CIV

RIG-I functions as a virus sensor that induces a cellular antiviral response. Although it has been investigated in other species, there have been no further studies to date on canine RIG-I against canine influenza virus (CIV). In the present study, we cloned the RIG-I gene of beagle dogs and characterized its expression, subcellular localization, antiviral response, and interactions with CIV proteins. RIG-I was highly expressed and mainly localized in the cytoplasm, with low levels detected in the nucleus. The results revealed that overexpression of the CARD domain of RIG-I and knockdown of RIG-I showed its ability to activate the RLR pathway and induced the expression of downstream interferon-stimulated genes. Moreover, overexpression of canine RIG-I suppressed the replication of CIV. The association between RIG-I and CIV was evaluated with the luciferase assay and by indirect immunofluorescence and bimolecular fluorescence complementation analyses. The results showed that CIV nonstructural protein 1 (NS1) can strongly suppress the RIG-I–mediated innate immune response, and the novel interactions between CIV matrix proteins (M1 and M2) and canine RIG-I were disclosed. These findings provide a basis for investigating the antiviral mechanism of canine RIG-I against CIV, which can lead to effective strategies for preventing CIV infection in dogs.

Investigation of Dengue Fever Outbreak in a Rural Area of Islamabad, Pakistan: A Case Control Study

Background: In the second week of October 2019, five suspected cases of dengue fever were reported from union council Sohan, Islamabad rural (population 45,747) to the health department, Islamabad Capital Territory (ICT). Outbreak investigation was conducted with the objectives to identify risk factors and to recommend control measures. Methods: Outbreak investigation was conducted from 17 th October to 25 th November 2019. A case was defined as, “fever and two or more of the following signs/symptoms; headache, retro-orbital pain, joint/bone pain, myalgia and petechial rash with NS1 test (Nonstructural Protein 1) positive during 8 th October to 25 th November 2019 among residents of Sohan”. Age and sex-matched controls were recruited from the same neighborhood. All cases were positive for NS1 antigen. Blood samples from five suspected cases were collected and tested for laboratory confirmation. Results: A total of 547 households were surveyed and 85 cases were identified. The mean age was 34.4 years + 16.05 (range 3-71 years). The attack rate was 0.19% whereas the most affected age group was the 45-54 years (AR 0.43%). Males were predominantly affected (n=48 56.5%). Among all cases, 32% (n=27)) had stagnant water inside or around their houses (aOR 2.65, CI 1.20-5.83, P= 0.005), 33% (n=28) were using mosquito repellent (aOR 0.35, CI 0.17-0.70, P <0.001), 31% (n=26) used indoor residual spray insecticide (aOR 0.48, CI 0.24-0.97, P =0.041), and 73% (n=62) used full protective clothing (aOR 0.17, CI 0.05-0.58, P <0.001). All five blood samples were tested positive for NS-1 antigen. Conclusion: The presence of accumulated rainwater in pools and empty receptacles around houses acted as breeding grounds for Aedes aegypti mosquitos and was the most probable cause of this outbreak. Following our recommendations, the health department-initiated mosquito breeding sites control activities through residual insecticide spray and advocacy on the use of protective measures against mosquito bites.