Background & Aims: Intrahepatic cholangiocyte organoids (ICOs) model was evaluated for host differences in HBV infection, cellular responses, antiviral and immunomodulator responses. Methods: 12 ICOs generated from liver resections and biopsies were assessed for metabolic markers and functional HBV entry receptor expression throughout differentiation. Structural changes relevant to HBV infection were characterised using histology, confocal and electron microscopy examinations. Optimal ICO culture conditions for HBV infection using HepAD38 (genotype D) and plasma derived HBV (genotype B & C) were described. HBV infection was confirmed using HBcAg immunostaining, qRT-PCR (RNA, cccDNA, extracellular DNA) and ELISA (HBsAg and HBeAg). Drug response to antiviral and immunosuppressive agent, and cellular responses (interferon-stimulated genes (ISG)) to interferon-α and viral mimic (PolyI:C) were assessed. Results: ICOs underwent metabolic and structural remodeling following differentiation. Optimal HBV infection was achieved in well-differentiated ICOs using spinoculation, with time and donor dependent increase in HBV RNA, cccDNA, extracellular DNA, HBeAg and HBsAg. Donor dependent drug-responsiveness to entry inhibitor and JAK inhibitor was observed. Despite having a robust ISG response to interferon-α and PolyI:C, HBV infection in ICOs did not upregulate ISGs. Conclusions: Human ICOs support HBV infection and replication with donor dependent variation in viral dynamics and cellular responses. These features can be utilised for development of personalised drug testing platform for antivirals.

In May 2022, a cluster of non-travel-related cases of human mpox were reported in the UK. The outbreak has since spread world-wide infecting over 85,000 patients and causing over 100 deaths. Recent data clearly suggest that patients infected with Human Immunodeficiency Virus (HIV) with CD4 counts less than 200 cells per mm 3 suffer significantly worse outcomes than immunocompetent patients. The available countermeasures lack robust clinical data and are deployed based on in vitro and animal studies as well as extrapolations from use against other poxviruses. In many cases, despite administration of these available treatments, initiation of antiretroviral therapy, and management of Immune Reconstitution Inflammatory Syndrome (IRIS), patients die. This review summarizes available data, identifies knowledge gaps and proposes recommendations on the management of severe mpox in PLWH.

Title: Interstitial Lung Diseases and COVID19 Pneumonia

Background: Human papillomaviruses (HPV) of the genus Betapapillomavirus can infect both cutaneous and mucosal sites, but research on its natural history at mucosal sites remains scarce. We examined the risk factors and co-detection patterns of HPVs of the Betapapillomavirus and Alphapapillomavirus genera in cervical samples of the Ludwig-McGill cohort study. Methods: We assessed a subset of 505 women from the Ludwig-McGill cohort study from São Paulo, Brazil. Cervical samples over the first year of follow-up were tested for DNA of over 40 alphapapillomavirus types and 43 betapapillomavirus types using a type-specific multiplex genotyping PCR assay. We assessed the risk factors for prevalent and incident betapapillomavirus type detection, and whether types were detected more frequently together than expected assuming independence using permutation tests, logistic regression, and Cox regression. Results: We observed significant within-genus clustering but not cross-genus clustering. Multiple betapapillomavirus types were co-detected in the same sample 2.24 (95%CI: 1.65-3.29) times more frequently than expected. Conversely, co-detections of alphapapillomavirus and betapapillomavirus types in the same sample occurred only 0.64 (95%CI: 0.51-0.83) times as often as expected under independence. In prospective analyses, positivity to one HPV genus was associated with a non-significant lower incidence of detection of types in the other genus. Lifetime number of sex partners and new sex partner acquisition were associated with lower risks of prevalent and incident betapapillomavirus detection. Conclusion: Betapapillomaviruses are commonly found in the cervicovaginal tract. Results suggest potentially different mechanisms of transmission for betapapillomavirus genital infections other than vaginal sex.

Background Understanding the cellular host factors that promote and inhibit viral entry is important for identifying viral countermeasures. CRISPR whole genome screens can be used to rapidly discover host factors that contribute to or impair viral entry. However, when using the live viruses and cellular lethality for selection, these screens can identify an overwhelming number of genes without specificity for the stage of the viral infection cycle. New screening methods are needed to identify host machinery contributing to specific steps of viral infection. Here, we developed a CRISPR whole genome screen and counter screen strategy based on a pseudoviral platform that allowed identification of genes specific to SARS-CoV-2 spike and vesicular stomatitis virus glycoprotein VSV-G mediated entry. Methods To focus the screen onto the entry step, we used non-lytic fluorescent reporters in combination with a comparative counter screen strategy to distinguish host genes affecting the pseudoviral reporter from those unique to envelope-mediated entry. Screening of SARS-CoV-2 spike and VSV-G on the same lentiviral pseudovirus allowed identification of entry-specific genes relative to genes associated with retro-transcription, integration, and reporter expression from the lentiviral pseudovirus. Second, a Cre-Gag fusion protein in the pseudovirus was used to bypass retro-transcription and integration by directly activating a floxed GFP reporter upon entry to reduce the number of gene hits and increase specificity for viral entry. Results Our approach correctly identified SARS-CoV-2 and VSV-G receptors ACE2 and LDLR, respectively and distinguished genes associated with retroviral reporter expression from envelope-mediated entry. Moreover, the CRE-Gag fusion/flox reporter increased the screen specificity for viral entry associated genes. Validation of a few hits demonstrates that this approach distinguishes envelope-specific host factors from genes affecting reporter expression. Conclusion Overall, this approach provides a new strategy for identifying host genes influencing viral entry without the confounding complexity of live-viral screens which produce long gene lists associated with all aspects of viral pathogenesis and replication. This approach provides a pathway for increasing the specificity of CRISPR whole genome screens for identifying host genes contributing to specific steps in viral infection.

Background: The introduction of primary HPV cervical cancer screening requires the implementation of an appropriate triage strategy that will be effective in detecting high-grade cervical disease without losing diagnostic specificity. Methods: From the 30.066 screening tests results, a total of 1086 with available high-risk human papillomavirus (HRHPV) with limited genotyping, cytology and p16/Ki67 dual-stain were selected. Two triage strategies for primary HPV screening were analyzed retrospectively based on the study group. Performance characteristics for p16/Ki67 and cytology triage in detection of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and grade 3 or worse (CIN3+) were calculated, detected in colposcopic biopsy. Results: In HPV16/18-positive cases, primary HPV with p16/Ki67 triage was significantly more specific than cytology (53.1%/16.8% for CIN2+; p<0.0001; 45.9%/17.0% for CIN3+; p<0.0001), with yielded sensitivity (95.7%/84.8% for CIN2+; p=0.0955; 100.0%/87.5% for CIN3+; p=0.0832). In other HRHPV-positive cases (N16/N18), p16/Ki67 triage was also significantly higher specific (51.3%/15.3% for CIN2+; p<0.0001; 44.5%/16.5% for CIN3+; p<0.0001), with sensitivity (92.3%/74.4% for CIN2+; p=0.0522; 90.9%/81.8% for CIN3+; p=0.5637). Diagnostic predictive values were significantly higher for p16/Ki67 triage with the highest PPV in HPV16/18-positive cases for CIN2+ (45.4%; 95% CI: 35.2-55.8; p<0.0001) and very high NPV in all HPV-positive cases regardless of detected genotype (96.3%-100.0%). The risk (1-NPV) for CIN3+ in HRHPV16/18-positive/p16/Ki67-negative women was 0.0%. Conclusions: Superior diagnostic performance compared to cytology for detecting cervical cancer precursors indicates that p16/Ki67 dual-immunostain may be a highly effective tool of triage in primary HPV screening with limited HPV 16/18 genotyping in the secondary cervical cancer prevention.

Purpose Although monoclonal antibodies specific to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are known, information about the B-cell receptor (BCR) repertoire and its change in patients during COVID-19 disease progression is underreported. Methods We used immunoglobulin heavy chain (IGH) variable region (IGHV) spectratyping and next-generation sequencing of peripheral blood B-cell genomic DNA collected at multiple time points during disease evolution to study B-cell response to SARS-CoV-2 infection in 14 individuals with acute COVID-19. Results We found a broad distribution of responding B-cell clones. The IGH gene usage was not significantly skewed but frequencies of individual IGH genes changed repeatedly. We found predominant usage of unmutated and low mutation-loaded IGHV rearrangements characterizing naïve and extrafollicular B-cells among the majority of expanded peripheral B-cell clonal lineages at most tested time points in most patients. IGH rearrangement usage showed no apparent relation to anti-SARS-CoV-2 antibody titers. Some patients demonstrated mono/oligoclonal populations carrying highly mutated IGHV rearrangements indicating antigen experience at some of the time points tested, including even before anti-SARS-CoV-2 antibodies were detected. Conclusion We present evidence demonstrating that the B-cell response to SARS-CoV-2 is individual and includes different lineages of B-cells at various time points during COVID-19 progression.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) enters the host cell by binding to angiotensin-converting enzyme 2 (ACE2). While evolutionarily conserved, ACE2 glycoproteins differ across various species and differential interactions with Spike (S) glycoproteins of SARS-CoV-2 viruses impact species specificity. Reverse zoonoses led to SARS-CoV-2 outbreaks on multiple American mink ( Mustela vison) farms during the pandemic and gave rise to mink-associated S substitutions known for transmissibility between mink and zoonotic transmission to humans. In this study, we used bio-layer interferometry (BLI) to discern the differences in binding affinity between multiple human and mink-derived S glycoproteins of SARS-CoV-2 and their respective ACE2 glycoproteins. Further, we conducted a structural analysis of a mink variant S glycoprotein and American mink ACE2 (mvACE2) using cryo-electron microscopy (cryo-EM), revealing four distinct conformations. We discovered a novel intermediary conformation where the mvACE2 glycoprotein is bound to the receptor-binding domain (RBD) of the S glycoprotein in a “down” position, approximately 34° lower than previously reported “up” RBD. Finally, we compared residue interactions in the S-ACE2 complex interface of S glycoprotein conformations with varying RBD orientations. These findings provide valuable insights into the molecular mechanisms of SARS-CoV-2 entry.

A document by Haleema Qayyum Abbasi. Click on the document to view its contents.

Limited antibody response after BA.4/5 adapted booster vaccination in rheumatic patients receiving anti-TNF therapy: results of a case seriesUlf Martin Geisen1*, Mathias Voß2*, Ruben Rose2*, Franziska Neumann3, Carina Bäumler3, Sina Müller3, Lea Paltzow3, Christina Martínez Christophersen3, Merle Münier3, Elena Hildebrand1, Paula Hoff4, Ann Carolin Longardt5, Thomas Lorentz3, Jan Henrik Schirmer1, Melike Sümbül6, Florian Tran7,8, Dennis Berner1, Helmut Fickenscher2, Sascha Gerdes6, Stefan Schreiber7,8, Andi Krumbholz2,3‡, Bimba Franziska Hoyer1‡1 Medical Department I, Rheumatology, and Clinical Immunology, Christian-Albrecht University of Kiel and University Medical Center Schleswig-Holstein Campus Kiel, Kiel, Germany2 Institute for Infection Medicine, Christian-Albrecht University of Kiel and University Medical Center Schleswig-Holstein, Campus Kiel, Kiel, Germany3 Labor Dr. Krause und Kollegen MVZ GmbH, Kiel, Germany4 Department of Rheumatology, Endokrinologikum-Gruppe, Berlin, Germany5 Department of Pediatrics, Christian-Albrecht University of Kiel and University Medical Center Schleswig-Holstein Campus Kiel, Kiel, Germany6 Department for Dermatology, Christian-Albrecht University of Kiel and University Medical Center Schleswig-Holstein Campus Kiel, Kiel, Germany7 Institute of Clinical Molecular Biology, Christian-Albrecht University of Kiel, Kiel, Germany8 Department for Internal Medicine I, Christian-Albrecht University of Kiel and University Medical Center Schleswig-Holstein Campus Kiel* ‡These authors contributed equallyCorresponding Author: Bimba Franziska HoyerMedical Department I, Rheumatology and Clinial Immunology,University Medical Center Schleswig-HolsteinArnold-Heller-Str. 324105 KielEmail: [email protected]: +49 431 500 22203

The use of adeno-associated virus (AAV) vectors in gene therapy has demonstrated great potential in treating genetic disorders. However, infusion-associated reactions (IARs) pose a significant challenge to the safety and efficacy of AAV-based gene therapy. This review provides a comprehensive summary of the current understanding of IARs to AAV therapy, including their underlying mechanisms, clinical presentation, and treatment options. Toll-like receptor activation and subsequent production of pro-inflammatory cytokines are associated with IARs, stimulating neutralizing antibodies and T-cell responses that interfere with gene therapy. Risk factors for IARs include high titers of pre-existing neutralizing antibodies, previous exposure to AAV, and specific comorbidities. Clinical presentation ranges from mild flu-like symptoms to severe anaphylaxis and can occur during or after AAV administration. There are no established guidelines for pre- and post-administration tests for AAV therapies, and routine laboratory requests are not standardized. Treatment options include corticosteroids, plasmapheresis, and supportive medications such as antihistamines and acetaminophen, but there is no consensus on the route of administration, dosage, and duration. This review highlights the inadequacy of current treatment regimens for IARs and the need for further research to improve the safety and efficacy of AAV-based gene therapy.

The main proteases (M pro) are highly conserved cysteine-rich proteins that can be covalently modified by numerous natural and synthetic compounds. Herein, we constructed an integrative approach to efficiently discover covalent inhibitors of M pro from complex herbal matrices. This work begins with biological screening of sixty clinically used antiviral herbal medicines, among which Lonicera japonica (LJ) demonstrated the strongest anti-M pro effect (IC 50 = 37.82 μg/mL). Mass spectrometry-based chemical analysis and chemoproteomic profiling revealed that LJ extract contains at least 50 constituents, of which 22 exhibited the capability to covalently modify M pro. We subsequently verified the anti-M pro effects of these covalent binders. Gallic acid and quercetin were found to potently inhibit SARS-CoV-2 M pro in dose- and time- dependent manners, with the IC 50 values below 10 µM. The inactivation kinetics, binding affinity and binding mode of gallic acid and quercetin were further characterized by fluorescence resonance energy transfer, surface plasmon resonance, and covalent docking simulations. Overall, this study established a practical approach for efficiently discovering the covalent inhibitors of M pro from herbal medicines by integrating target-based high-throughput screening and mass spectrometry-based assays, which would greatly facilitate the discovery of key anti-viral constituents from medicinal plants.

A document by Haleema Qayyum Abbasi. Click on the document to view its contents.

During the Belgian winter and spring season 2022-2023, we investigated the potential of used paper tissue (UPT) as a non-invasive sampling method for the diagnosis of acute respiratory infections. Screening for respiratory pathogens was done using an in-house developed respiratory panel for simultaneous detection of 22 respiratory viruses and 7 non-viral pathogens. The method allowed the identification and typing of respiratory pathogens in symptomatic individuals, as well as in collective samples taken at a community level. Pathogens that were identified in nasal swabs could also be detected in concurrent UPT from the same patient. In all cases that tested positive on an antigen-detection rapid diagnostic test, the corresponding virus could be detected in UPT. The collection of UPT could be useful in epidemiological surveillance of SARS-CoV-2 and other coronaviruses, as well as other respiratory pathogens such as influenzavirus, respiratory syncytial virus, entero/rhinoviruses including EV D68, parainfluenzaviruses and Streptococcus pneumoniae. Multiple respiratory pathogens could be detected in UPTs of collectivities, confirming its applicability for community testing. This is especially interesting for screening in nursing homes, centers for the disabled, schools or other settings were taking nasal or nasopharyngeal samples is cumbersome.

The number of SARS-CoV-2 recombinants identified during the pandemic has increased since the era of Omicron variants, but XBB.1.5 (or Omicron 23A) is the first lineage comprised of hybrid genomes to predominate at the country and global scales. Very interestingly, the XBB.1.5 recombinant, like the Marseille-4B subvariant (B.1.160/20A.EU2) and the pandemic variant B.1.1.7 (20I/Alpha) previously, has its ORF8 gene inactivated by a stop codon. XBB.1.5 was generated through two successive main events: a recombination between SARS-CoV-2 of lineages BA.2.10.1.1 (BJ.1) and BA.2 75.3.1.1.1 (BM.1.1.1) that generated the XBB (22F) lineage; then ORF8 gene inactivation by a stop codon. We further identified that a stop codon was present at 89 (74%) codons of the ORF8 gene in ≥1 of 15,222,404 genomes available in GISAID, and at 15 codons (12%) in ≥1,000 genomes. Thus, it is very likely that stop codons in ORF8 gene contributed on at least 3 occasions and independently during the SARS-CoV-2 pandemic to the evolutionary success of a lineage that became transiently predominant, most recently XBB.1.5. Such association of gene loss with evolutionary success, which suits the recently described Mistigri rule, is an important biological phenomenon very unknown in virology while largely described in cellular organisms.

The monkeypox virus (MPXV) outbreak in 2022 has renewed interest in the detection of antibodies against orthopox viruses (OPXV) and MPXV, as serological methods can aid diagnostics and are key to epidemiological studies. Here three complementary serological methods are described with different strengths to aid the development and evaluation of in-house assays: An immunofluorescence assay (IFA) for specific detection of IgG and IgM, an enzyme-linked immunosorbent assay (ELISA) for higher sample throughput to aid epidemiological studies and a neutralization test (NT) to detect virus neutralising antibodies. As implementation of MPXV-specific diagnostics is often hampered by the requirement for a dedicated biosafety level 3 laboratory (BSL-3), the focus is on biosafety aspects to facilitate safe testing also under BSL-2 conditions. To this aim, IFA and NT titres of sera are compared after MPXV or cowpox virus infection or vaccinia virus immunization by using both homologous and heterologous antigens. Because of a large degree of cross-reactivity, vaccinia virus could be used as a less virulent alternative to MPXV, as VACV can be handled under BSL-2 conditions. Furthermore, an inactivation method to remove potentially infectious virus particles from viraemic sera without compromising antibody detection was developed. Although the existing immunological cross-reactivity between OPXV prevents species differentiation by general serological methods, it also provides an opportunity for the interchangeable usage of different OPXV species in serological assays, enabling MPXV serology outside of BSL-3 facilities.

Evaluate clinical effectiveness of Azvudine with data rather than speculationDaishi Li, MD1,2,3*, Yihuang Liu, MD1,2,3*, Minxue Shen, MD1,2,3,4#, Guangtong Deng, MD1,2,3#1 Department of Dermatology, Hunan Engineering Research Center of Skin Health and Disease, Hunan Key Laboratory of Skin Cancer and Psoriasis, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China2 National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China3 Furong Laboratory, Xiangya Hospital, Central South University, Changsha, Hunan, China.4 Department of Social Medicine and Health Management, Xiangya School of Public Health, Central South University, Changsha, Hunan, ChinaDaishi Li and Yihuang Liu contributed equally to this study.#Correspondence to Guangtong Deng, Minxue Shen, MD, Department of Dermatology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China. E-mail: [email protected]; [email protected] the Editor,We appreciate the interest in our article entitled “Real-world effectiveness of Azvudine versus nirmatrelvir-ritonavir in hospitalized patients with COVID-19: A retrospective cohort study”.In their study, Ma et al. stated that antiviral therapies must be administered within five days of symptom onset to be effective. However, our study population included approximately 90% of patients beyond this treatment window. Moreover, we were unable to provide virological results, leading to Ma et al.’s speculation that treatment was unlikely to benefit patients in our study. They further praised the benefits of nirmatrelvir-ritonavir in COVID-19 patients and demonstrated their results supporting a faster time to nucleic acid negative conversion compared to patients receiving Azvudine. In a phase III trial, patients treated with Azvudine only showed a 50% reduction in viral load on the fifth day of treatment, making it difficult to achieve significant clinical efficacy. Ma et al. also concluded that the dose of Azvudine was insufficient for its antiviral effects, using data from drug experimental and pharmacokinetic studies. Finally, they called for the time to make more effective drugs available to COVID-19 patients.1As a retrospective study, we sought to collect more information, particularly on cycle thresholds in COVID-19 patients. However, as we previously reported, the cycle threshold value was no longer used as a discharge criterion during the period and was not regularly checked.2 Moreover, most electronic health records of COVID-19 patients only recorded quantitative results (positive or negative) upon admission, hence virological data was lacking. Nevertheless, it was not necessary for our study, which aimed to evaluate the real-world clinical effectiveness (composite outcomes and mortality). 3Azvudine and nirmatrelvir-ritonavir were recommended for the treatment of COVID-19 patients as early as possible, but these drugs were not strictly used according to the instructions during the special period. We acknowledged several limitations in our retrospective study, including the possibility of selection bias and confounding by indication. We also emphasized that our conclusions were solely based on data from our hospital and that the generalization of our findings required more high-quality and multi-center clinical trials.3 As clinicians, with limited knowledge about drug experimental and pharmacokinetics studies, we refrain from making any response on the topic of its antiviral effect. If there are any questions concerning the antiviral effect, we suggest contacting the corresponding authors of these studies.4,5 We can, however, discuss the clinical effectiveness of the drugs.We agree that numerous studies from other countries support the benefits of nirmatrelvir-ritonavir in treating COVID-19 patients.6 However, a multi-center randomized controlled study based on Chinese patients failed to detect a significant reduction in the risk of all-cause mortality on day 28 and the duration of virus clearance in severe adult COVID-19 patients.7 As our study mainly included severe COVID-19 patients in China, the limited efficacy of nirmatrelvir-ritonavir is not surprising.3,8 While Ma et al. found a faster time to nucleic acid negative conversion in patients receiving nirmatrelvir-ritonavir compared to those receiving Azvudine,1 it did not entirely negate our findings that support the better clinical benefit of Azvudine.3Evaluating a drug’s effectiveness requires clinical data, rather than piecing together several basic articles to make a speculation solely based on its antiviral effect. For instance, remdesivir can effectively inhibit COVID-19 infection in vitro but has no significant effect on COVID-19 patients who are already being ventilated.9Similarly, metformin, the most commonly used oral type 2 diabetic drug, functions in many diseases, including avoiding long COVID.10 Nowadays, compared with other anti-COVID-19 drugs, there is limited clinical study on Azvudine. Therefore, we call for more real-world studies to evaluate the effectiveness of Azvudine in COVID-19 patients.Finally, we appreciate the feedback from some readers regarding the flowchart of patient screening in Figure 1. To avoid any confusion, we would like to clarify that patients were repeatedly counted if they met individual exclusion criteria in the study. Additionally, pregnant patients were mistakenly classified to patients with history diseases. After matching, none of pregnant patients were included in Azudine and nirmatrelvir-ritonavir group. Therefore, our conclusions were still consistent.Declaration of Competing Interest: None.Acknowledgement: This work was supported by the National Natural Science Foundation of China (Grant Nos. 82102803, 82272849 to G. D.), National Natural Science Foundation of Hunan Province (Grant Nos. 2021JJ40976 to G. D.) and Natural Science Fund for Outstanding Youths in Hunan Province (2023JJ20093 to G.D.).Role of the funding source: The funding sources had no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the article for publication. All the authors had full access to all the data in the study and agree to submit the manuscript for publication.

SARS-CoV2 infection has caused an increase in mortality and morbidity but with vaccination, the disease severity has significantly reduced. But with emergence of various variants of concerns (VOCs), the vaccine breakthrough infection has also increased. Here, we studied circulating spike specific T follicular response (cTfh) in infection naïve vaccinees and convalescent vaccinees (individuals who got delta breakthrough infection after 2 doses of BBV152 vaccine) to understand their response as they are the most crucial cells that are involve in vaccine mediated protection by helping in B-cell maturation. Our results indicated that cTfh cells in both the groups recognized wild type and delta spike protein but memory response to wild type spike was superior in infection naïve than in convalescent group. The cytokine response particularly IL-21 from cTfh cells was also higher in infection naïve than convalescent vaccines indicating a dampened cTfh response in convalescent vaccines after breakthrough infection. Also, there was positive correlation between IL-21 from cTfh cells and neutralizing antibodies of infection naïve vaccinees. Multiple cytokine analysis also revealed higher inflammation in convalescent vaccinees. Our data indicated necessity of third booster dose may be individual specific depending in the steady state functional phenotype of immune cells.