Personal historical perspective of HIV: part 11.

All animal life on earth is thought to have a common origin and have common genetic mechanisms. Evolution has enabled differentiation of species. Pathogens likewise have evolved within various species and mostly come to a settled dynamic equilibrium such that co-existence results (pathogens ideally should not kill their hosts). Problems arise when pathogens jump species because the new host had not developed any resistance. These infections from related species are known as zoonoses. COVID-19 is the latest example of a virus entering another species but HIV (and various strains of influenza) were previous examples. HIV entered the human population from monkeys in Africa. These two papers outline the underlying principle of HIV and the differing epidemiologies in Africa, the USA and in Edinburgh. The underlying immunosuppression of HIV in Africa was initially hidden behind common infections and HIV first came to world awareness in focal areas of the USA as a disease seemingly limited to gay males. The epidemic of intravenous drug abuse in Edinburgh was associated with overlapping epidemics of bloodborne viruses like hepatitis B, hepatitis C and HIV.

The preference signature of the SARS-CoV-2 Nucleocapsid NTD for its 5'-genomic RNA elements.

The nucleocapsid protein (N) of SARS-CoV-2 plays a pivotal role during the viral life cycle. It is involved in RNA transcription and accounts for packaging of the large genome into virus particles. N manages the enigmatic balance of bulk RNA-coating versus precise RNA-binding to designated cis-regulatory elements. Numerous studies report the involvement of its disordered segments in non-selective RNA-recognition, but how N organizes the inevitable recognition of specific motifs remains unanswered. We here use NMR spectroscopy to systematically analyze the interactions of N's N-terminal RNA-binding domain (NTD) with individual cis RNA elements clustering in the SARS-CoV-2 regulatory 5'-genomic end. Supported by broad solution-based biophysical data, we unravel the NTD RNA-binding preferences in the natural genome context. We show that the domain's flexible regions read the intrinsic signature of preferred RNA elements for selective and stable complex formation within the large pool of available motifs.

Novel SARS-CoV-2 entry inhibitors, 2-anilinoquinazolin-4(3H)-one derivatives, show potency as SARS-CoV-2 antivirals in a human ACE2 transgenic mouse model.

The ongoing COVID-19 has not only caused millions of deaths worldwide, but it has also led to economic recession and the collapse of public health systems. The vaccines and antivirals developed in response to the pandemic have improved the situation markedly; however, the pandemic is still not under control with recurring surges. Thus, it is still necessary to develop therapeutic agents. In our previous studies, we designed and synthesized a series of novel 2-anilinoquinazolin-4(3H)-one derivatives, and demonstrated inhibitory activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and MERS-CoV in vitro. We then conducted in vivo studies using modified compounds that are suitable for oral administration. These compounds demonstrated no toxicity in rats and inhibited viral entry. Here, we investigated the in vivo efficacy of these drug candidates against SARS-CoV-2. Three candidate drugs, 7-chloro-2-((3,5-dichlorophenyl)amino)quinazolin-4(3H)-one (1), N-(7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-N-(3,5-dichlorophenyl)acetamide (2), and N-(7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-N-(3,5-difluorophenyl)acetamide (3) were administered orally to hACE2 transgenic mice at a dose of 100 mg/kg. All three drugs improved survival rate and reduced the viral load in the lungs. These results show that the derivatives possess in vivo antiviral efficacy similar to that of molnupiravir, which is currently being used to treat COVID-19. Overall, our data suggest that 2-anilinoquinazolin-4(3H)-one derivatives are promising as potential oral antiviral drug candidates against SARS-CoV-2 infection.

Spatial genetic structure of 2009 H1N1 pandemic influenza established as a result of interaction with human populations in mainland China.

Identifying the spatial patterns of genetic structure of influenza A viruses is a key factor for understanding their spread and evolutionary dynamics. In this study, we used phylogenetic and Bayesian clustering analyses of genetic sequences of the A/H1N1pdm09 virus with district-level locations in mainland China to investigate the spatial genetic structure of the A/H1N1pdm09 virus across human population landscapes. Positive correlation between geographic and genetic distances indicates high degrees of genetic similarity among viruses within small geographic regions but broad-scale genetic differentiation, implying that local viral circulation was a more important driver in the formation of the spatial genetic structure of the A/H1N1pdm09 virus than even, countrywide viral mixing and gene flow. Geographic heterogeneity in the distribution of genetic subpopulations of A/H1N1pdm09 virus in mainland China indicates both local to local transmission as well as broad-range viral migration. This combination of both local and global structure suggests that both small-scale and large-scale population circulation in China is responsible for viral genetic structure. Our study provides implications for understanding the evolution and spread of A/H1N1pdm09 virus across the population landscape of mainland China, which can inform disease control strategies for future pandemics.

Effectiveness of regdanvimab treatment for SARS-CoV-2 delta variant, which exhibited decreased in vitro activity: a nationwide real-world multicenter cohort study.

Background: Immune-evading severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are emerging continuously. The clinical effectiveness of monoclonal antibody agents that exhibit decreased in vitro activity against SARS-CoV-2 variants needs to be elucidated. Methods: A nationwide, multicenter, retrospective cohort study was designed to evaluate the effectiveness of regdanvimab, an anti-SARS-CoV-2 monoclonal antibody agent. Regdanvimab was prescribed in South Korea before and after the emergence of the delta variant, against which the in vitro activity of regdanvimab was decreased but present. Mild to moderate coronavirus 2019 (COVID-19) patients with risk factors for disease progression who were admitted within seven days of symptom onset were screened in four designated hospitals between December 2020 and September 2021. The primary outcomes, O2 requirements and progression to severe disease within 21 days of admission, were compared between the regdanvimab and supportive care groups, with a subgroup analysis of delta variant-confirmed patients. Results: A total of 2,214 mild to moderate COVID-19 patients were included, of whom 1,095 (49.5%) received regdanvimab treatment. In the analysis of the total cohort, significantly fewer patients in the regdanvimab group than the supportive care group required O2 support (18.4% vs. 27.1%, P < 0.001) and progressed to severe disease (4.0% vs. 8.0%, P < 0.001). In the multivariable analysis, regdanvimab was significantly associated with a decreased risk for O2 support (HR 0.677, 95% CI 0.561-0.816) and progression to severe disease (HR 0.489, 95% CI 0.337-0.709). Among the 939 delta-confirmed patients, O2 support (21.5% vs. 23.5%, P = 0.526) and progression to severe disease (4.2% vs. 7.3%, P = 0.055) did not differ significantly between the regdanvimab and supportive care groups. In the multivariable analyses, regdanvimab treatment was not significantly associated with a decreased risk for O2 support (HR 0.963, 95% CI 0.697-1.329) or progression to severe disease (HR 0.665, 95% CI 0.349-1.268) in delta-confirmed group. Conclusions: Regdanvimab treatment effectively reduced progression to severe disease in the overall study population, but did not show significant effectiveness in the delta-confirmed patients. The effectiveness of dose increment of monoclonal antibody agents should be evaluated for variant strains exhibiting reduced susceptibility.

A viral pan-end RNA element and host complex define a SARS-CoV-2 regulon.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, generates multiple protein-coding, subgenomic RNAs (sgRNAs) from a longer genomic RNA, all bearing identical termini with poorly understood roles in regulating viral gene expression. Insulin and interferon-gamma, two host-derived, stress-related agents, and virus spike protein, induce binding of glutamyl-prolyl-tRNA synthetase (EPRS1), within an unconventional, tetra-aminoacyl-tRNA synthetase complex, to the sgRNA 3'-end thereby enhancing sgRNA expression. We identify an EPRS1-binding sarbecoviral pan-end activating RNA (SPEAR) element in the 3'-end of viral RNAs driving agonist-induction. Translation of another co-terminal 3'-end feature, ORF10, is necessary for SPEAR-mediated induction, independent of Orf10 protein expression. The SPEAR element enhances viral programmed ribosomal frameshifting, thereby expanding its functionality. By co-opting noncanonical activities of a family of essential host proteins, the virus establishes a post-transcriptional regulon stimulating global viral RNA translation. A SPEAR-targeting strategy markedly reduces SARS-CoV-2 titer, suggesting a pan-sarbecoviral therapeutic modality.

Sustainable access of quality seeds of genetically engineered crops in Eastern Africa - Case study of Bt Cotton.

The genetically engineered bollworm-resistant Bt cotton hybrid varieties offer opportunities for reducing crop losses and enhancing productivity. In Eastern Africa region, Sudan, Ethiopia, and Kenya have approved and released Bt cotton in 2012, in 2018, and in 2019, respectively. The region has potential to grow cotton in over 5 million hectares. For commercial plantings in Ethiopia, Sudan and Kenya, hybrid Bt cotton seeds have been imported from India. Due to the COVID-19 pandemic-induced supply chain disruptions, high shipment costs, bureaucratic procedures for importing seeds, and foreign exchange shortages, farmers have not been able to access Bt cotton seeds. Stakeholders are seeking local production of seeds to provide sustainable access by farmers at affordable cost. Country case studies reveal the importance of enhancing capacity for local seed production and extension advisory services. Revival of the cotton sector needs enhanced public-private partnerships to pave the way for sustainable seeds access in the region.

SARS-CoV-2 detection and inactivation in water and wastewater: review on analytical methods, limitations and future research recommendations.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been detected in wastewater. Wastewater-based epidemiology (WBE) is a practical and cost-effective tool for the assessment and controlling of pandemics and probably for examining SARS-CoV-2 presence. Implementation of WBE during the outbreaks is not without limitations. Temperature, suspended solids, pH, and disinfectants affect the stability of viruses in wastewater. Due to these limitations, instruments and techniques have been utilized to detect SARS-CoV-2. SARS-CoV-2 has been detected in sewage using various concentration methods and computer-aided analyzes. RT-qPCR, ddRT-PCR, multiplex PCR, RT-LAMP, and electrochemical immunosensors have been employed to detect low levels of viral contamination. Inactivation of SARS-CoV-2 is a crucial preventive measure against coronavirus disease 2019 (COVID-19). To better assess the role of wastewater as a transmission route, detection, and quantification methods need to be refined. In this paper, the latest improvements in quantification, detection, and inactivation of SARS-CoV-2 in wastewater are explained. Finally, limitations and future research recommendations are thoroughly described.

Low-Cost, Real-Time Polymerase Chain Reaction System with Integrated RNA Extraction.

Rapid, easy-to-use, and low-cost systems for biological sample testing are important for point-of-care diagnostics and various other health applications. The recent pandemic of Coronavirus Disease 2019 (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) showed an urgent need to rapidly and accurately identify the genetic material of SARS-CoV-2, an enveloped ribonucleic acid (RNA) virus, in upper respiratory specimens from people. In general, sensitive testing methods require genetic material extraction from the specimen. Unfortunately, current commercially available extraction kits are expensive and involve time-consuming and laborious extraction procedures. To overcome the difficulties associated with common extraction methods, we propose a simple enzymatic assay for the nucleic acid extraction step using heat mediation to improve the polymerase chain reaction (PCR) reaction sensitivity. Our protocol was tested on Human Coronavirus 229E (HCoV-229E) as an example, which comes from the large coronaviridae family of viruses that affect birds, amphibians, and mammals, of which SARS-CoV-2 is a member. The proposed assay was performed using a low-cost, custom-made, real-time PCR system that incorporates thermal cycling and fluorescence detection. It had fully customizable reaction settings to allow versatile biological sample testing for various applications, including point-of-care medical diagnosis, food and water quality testing, and emergency health situations. Our results show that heat-mediated RNA extraction is a viable extraction method when compared to commercial extraction kits. Further, our study showed that extraction has a direct impact on purified laboratory samples of HCoV-229E, but no direct impact on infected human cells. This is clinically relevant, as it allows us to circumvent the extraction step on clinical samples when using PCR.

The PRMT5/WDR77 complex restricts hepatitis E virus replication.

Hepatitis E virus (HEV) is one of the main pathogenic agents of acute hepatitis in the world. The mechanism of HEV replication, especially host factors governing HEV replication is still not clear. Here, using HEV ORF1 trans-complementation cell culture system and HEV replicon system, combining with stable isotope labelling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we aimed to identify the host factors regulating HEV replication. We identified a diversity of host factors associated with HEV ORF1 protein, which were putatively responsible for viral genomic RNA replication, in these two cell culture models. Of note, the protein arginine methyltransferase 5 (PRMT5)/WDR77 complex was identified in both cell culture models as the top hit. Furthermore, we demonstrated that PRMT5 and WDR77 can specifically inhibit HEV replication, but not other viruses such as HCV or SARS-CoV-2, and this inhibition is conserved among different HEV strains and genotypes. Mechanistically, PRMT5/WDR77 can catalyse methylation of ORF1 on its R458, impairing its replicase activity, and virus bearing R458K mutation in ORF1 relieves the restriction of PRMT5/WDR77 accordingly. Taken together, our study promotes more comprehensive understanding of viral infections but also provides therapeutic targets for intervention.

Delivery platforms for broadly neutralizing antibodies.

PURPOSE OF REVIEW: Passive administration of broadly neutralizing antibodies (bNAbs) is being evaluated as a therapeutic approach to prevent or treat HIV infections. However, a number of challenges face the widespread implementation of passive transfer for HIV. To reduce the need of recurrent administrations of bNAbs, gene-based delivery approaches have been developed which overcome the limitations of passive transfer. RECENT FINDINGS: The use of DNA and mRNA for the delivery of bNAbs has made significant progress. DNA-encoded monoclonal antibodies (DMAbs) have shown great promise in animal models of disease and the underlying DNA-based technology is now being tested in vaccine trials for a variety of indications. The COVID-19 pandemic greatly accelerated the development of mRNA-based technology to induce protective immunity. These advances are now being successfully applied to the delivery of monoclonal antibodies using mRNA in animal models. Delivery of bNAbs using viral vectors, primarily adeno-associated virus (AAV), has shown great promise in preclinical animal models and more recently in human studies. Most recently, advances in genome editing techniques have led to engineering of monoclonal antibody expression from B cells. These efforts aim to turn B cells into a source of evolving antibodies that can improve through repeated exposure to the respective antigen. SUMMARY: The use of these different platforms for antibody delivery has been demonstrated across a wide range of animal models and disease indications, including HIV. Although each approach has unique strengths and weaknesses, additional advances in efficiency of gene delivery and reduced immunogenicity will be necessary to drive widespread implementation of these technologies. Considering the mounting clinical evidence of the potential of bNAbs for HIV treatment and prevention, overcoming the remaining technical challenges for gene-based bNAb delivery represents a relatively straightforward path towards practical interventions against HIV infection.

Importance of the SARS-CoV-2 genome and spike protein in the immunopathogenesis of COVID-19 and in the efficacy of vaccines.

Coronavirus (CoV) infections cause respiratory and enteric diseases with clinical manifestations ranging from faint to severe, even lead to death of patients. High connectivity between nations and infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represent a global health problem as the coronavirus disease 19 (COVID-19). This CoV-2 that cause SARS, which appeared in Wuhan, China, in December 2019 originated COVID-19 and declared as pandemic a few months posterior its appearance. In this review, the genomic and spike protein characteristics of SARS-CoV-2, the role of SARS-CoV-2 in the COVID-19 pathogenesis, cytokine storm, the role of cytotoxic T and B cells against SARS-CoV-2, as well as the vaccines efficacy (taking into account mutations in the spike protein) are described.

Los coronavirus (CoV) causan enfermedades respiratorias y entéricas leves, graves o críticas, pudiendo ocasionar la muerte del paciente. Debido a la alta conectividad entre naciones y a la transmisión, actualmente la COVID-19 representa un verdadero problema de salud pública en todo el mundo. El CoV-2 causante del síndrome respiratorio agudo grave (SARS-CoV-2) apareció a finales de diciembre de 2019 en Wuhan, China, y en marzo de 2020 la COVID-19 fue declarada pandemia. En esta revisión se describen las características del genoma y de la proteína espiga del SARS-CoV-2, su papel en la inmunopatogénesis de la COVID-19, la tormenta de citocinas, la actividad citotóxica inducida por células T y la producción de anticuerpos contra el SARS-CoV-2 mediada por células B, así como la eficacia de algunas vacunas, tomando en cuenta las mutaciones presentes en la proteína espiga.

The clinical significance of macrolide resistance in pediatric Mycoplasma pneumoniae infection during COVID-19 pandemic.

Background: Mycoplasma pneumoniae (MP) is a commonly occurring pathogen causing community-acquired pneumonia (CAP) in children. The global prevalence of macrolide-resistant MP (MRMP) infection, especially in Asian regions, is increasing rapidly. However, the prevalence of MRMP and its clinical significance during the COVID-19 pandemic is not clear. Methods: This study enrolled children with molecularly confirmed macrolide-susceptible MP (MSMP) and MRMP CAP from Beijing Children's Hospital Baoding Hospital, Capital Medical University between August 2021 and July 2022. The clinical characteristics, laboratory findings, chest imaging presentations, and strain genotypes were compared between patients with MSMP and MRMP CAP. Results: A total of 520 hospitalized children with MP-CAP were enrolled in the study, with a macrolide resistance rate of 92.7%. Patients with MRMP infection exhibited more severe clinical manifestations (such as dyspnea and pleural effusion) and had a longer hospital stay than the MSMP group. Furthermore, abnormal blood test results (including increased LDH and D-dimer) were more common in the MRMP group (P<0.05). Multilocus variable-number tandem-repeat analysis (MLVA) was performed on 304 samples based on four loci (Mpn13-16), and M3562 and M4572 were the major types, accounting for 74.0% and 16.8% of the strains, respectively. The macrolide resistance rate of M3562 strains was up to 95.1%. Conclusion: The prevalence of MRMP strains in hospitalized CAP patients was extremely high in the Baoding area, and patients infected with MRMP strains exhibited more severe clinical features and increased LDH and D-dimer. M3562 was the predominant resistant clone.

Evaluation of the Inhibition Potency of Nirmatrelvir against Main Protease Mutants of SARS-CoV-2 Variants.

SARS-CoV-2 continues to pose a threat to public health. Main protease (Mpro) is one of the most lucrative drug targets for developing specific antivirals against SARS-CoV-2 infection. By targeting Mpro, peptidomimetic nirmatrelvir is able to inhibit viral replication of SARS-CoV-2 and reduce the risk for progression to severe COVID-19. However, multiple mutations in the gene encoding Mpro of emerging SARS-CoV-2 variants raise a concern of drug resistance. In the present study, we expressed 16 previously reported SARS-CoV-2 Mpro mutants (G15S, T25I, T45I, S46F, S46P, D48N, M49I, L50F, L89F, K90R, P132H, N142S, V186F, R188K, T190I, and A191V). We evaluated the inhibition potency of nirmatrelvir against these Mpro mutants and solved the crystal structures of representative Mpro mutants of SARS-CoV-2 bound to nirmatrelvir. Enzymatic inhibition assays revealed that these Mpro variants remain susceptible to nirmatrelvir as the wildtype. Detailed analysis and structural comparison provided the inhibition mechanism of Mpro mutants by nirmatrelvir. These results informed the ongoing genomic surveillance of drug resistance of emerging SARS-CoV-2 variants to nirmatrelvir and facilitate the development of next-generation anticoronavirus drugs.

A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application.

Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is the second leading cause of death after COVID-19 pandemic. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform for tuberculosis diagnosis, termed MTB-MCDA-CRISPR. MTB-MCDA-CRISPR pre-amplified the specific sdaA gene of MTB by MCDA, and the MCDA results were then decoded by CRISPR-Cas12a-based detection, resulting in simple visual fluorescent signal readouts. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the sdaA gene of MTB. The optimal temperature for MCDA pre-amplification is 67°C. The whole experiment process can be completed within one hour, including sputum rapid genomic DNA extraction (15 minutes), MCDA reaction (40 minutes), and CRISPR-Cas12a-gRNA biosensing process (5 minutes). The limit of detection (LoD) of the MTB-MCDA-CRISPR assay is 40 fg per reaction. The MTB-MCDA-CRISPR assay does not cross reaction with non-tuberculosis mycobacterium (NTM) strains and other species, validating its specificity. The clinical performance of MTB-MCDA-CRISPR assay was higher than that of the sputum smear microscopy test and comparable to that of Xpert method. In summary, the MTB-MCDA-CRISPR assay is a promising and effective tool for tuberculosis infection diagnosis, surveillance and prevention, especially for point-of-care (POC) test and field deployment in source-limited regions.

The coronavirus recombination pathway.

Recombination is thought to be a mechanism that facilitates cross-species transmission in coronaviruses, thus acting as a driver of coronavirus spillover and emergence. Despite its significance, the mechanism of recombination is poorly understood, limiting our potential to estimate the risk of novel recombinant coronaviruses emerging in the future. As a tool for understanding recombination, here, we outline a framework of the recombination pathway for coronaviruses. We review existing literature on coronavirus recombination, including comparisons of naturally observed recombinant genomes as well as in vitro experiments, and place the findings into the recombination pathway framework. We highlight gaps in our understanding of coronavirus recombination illustrated by the framework and outline how further experimental research is critical for disentangling the molecular mechanism of recombination from external environmental pressures. Finally, we describe how an increased understanding of the mechanism of recombination can inform pandemic predictive intelligence, with a retrospective emphasis on SARS-CoV-2.

A resource of human coronavirus protein-coding sequences in a flexible, multipurpose Gateway Entry clone collection.

The COVID-19 pandemic has catalyzed unprecedented scientific data and reagent sharing and collaboration, which enabled understanding the virology of the SARS-CoV-2 virus and vaccine development at record speed. The pandemic, however, has also raised awareness of the danger posed by the family of coronaviruses, of which 7 are known to infect humans and dozens have been identified in reservoir species, such as bats, rodents, or livestock. To facilitate understanding the commonalities and specifics of coronavirus infections and aspects of viral biology that determine their level of lethality to the human host, we have generated a collection of freely available clones encoding nearly all human coronavirus proteins known to date. We hope that this flexible, Gateway-compatible vector collection will encourage further research into the interactions of coronaviruses with their human host, to increase preparedness for future zoonotic viral outbreaks.

Poor neutralizing antibody responses against SARS-CoV-2 Omicron BQ.1.1 and XBB in Norway in October 2022.

New immune evasive variants of SARS-CoV-2 continue to emerge, potentially causing new waves of covid-19 disease. Here, we evaluate levels of neutralizing antibodies against isolates of Omicron variants, including BQ.1.1 and XBB, in sera harvested 3-4 weeks after vaccination or breakthrough infections. In addition, we evaluate neutralizing antibodies in 32 sera from October 2022, to evaluate immunity in Norwegian donors prior to the winter season. Most serum samples harvested in October 2022 had low levels of neutralizing antibodies against BQ.1.1 and especially XBB, explaining why these variants and their descendants have dominated in Norway during the 2022 and 2023 winter season.