The clinical presentation of Mpox (monkeypox) resembles that of several other sexually transmitted infections. Although cases of Mpox are thought to be overlooked in the clinical setting, no study has investigated whether this is the case in Japan. In this study, we retrospectively screened suspected cases of syphilis to investigate overlooked cases of Mpox in Japan. We retrospectively analyzed 88 saliva samples and 78 whole blood samples from 88 patients treated in general internal medicine and infectious disease clinics. Molecular diagnostic testing revealed Mpox virus DNA in the saliva and blood samples of 1 patient with suspected primary syphilis in April 2023. This case was retrospectively diagnosed as Mpox. The findings of this study indicate that health care providers should be aware of Mpox as a differential diagnosis in patients with sexually transmitted infections. Improved access to molecular diagnostic testing may reduce the risk of overlooked cases of Mpox. Retrospective screening using saliva samples could help to increase opportunities to intervene against Mpox transmission.

The COVID-19 pandemic has resulted in the loss of millions of lives, although a majority of those infected have managed to survive. Consequently, a set of outcomes, identified as long COVID, is now emerging. While the primary target of SARS-CoV-2 is the respiratory system, the impact of COVID-19 extends to various body parts, including the bone. This study aims to investigate the effects of acute SARS-CoV-2 infection on osteoclastogenesis, utilizing both ancestral and Omicron viral strains. Monocyte-derived macrophages (MDM), which serve as precursors to osteoclasts, were exposed to both viral variants. However, the infection proved abortive, even though ACE2 receptor expression increased post-infection, with no significant impact on cellular viability and redox balance. Both SARS-CoV-2 strains heightened osteoclast formation in a dose-dependent manner, as well as CD51/61 expression and bone resorptive ability. Notably, SARS-CoV-2 induced early pro-inflammatory M1 macrophage polarization, shifting towards an M2-like profile. Osteoclastogenesis-related genes (RANK, NFATc1, DC-STAMP, MMP9) were upregulated, and surprisingly, SARS-CoV-2 variants promoted RANKL-independent osteoclast formation. This thorough investigation illuminates the intricate interplay between SARS-CoV-2 and osteoclast precursors, suggesting potential implications for bone homeostasis and opening new avenues for therapeutic exploration in COVID-19.

We estimated the dynamics of the neutralizing response against XBB sublineages and T cell response in PWH with previous AIDS and/or CD4<200/mm 3 receiving the bivalent original strain/BA.4-5 booster dose (bBD) in fall 2022. Samples were collected before the shot (T0), 15 days (T1), 3 (T3), and 6 months (T6) after. PWH were stratified by immunization status: hybrid immunity (HI; vaccination plus COVID-19) vs. non-hybrid immunity (nHI; vaccination only). At T1, 16% and 30% of PWH were non-responders in terms of anti-XBB.1.16 or anti-EG.5.1 nAbs, respectively. At T3, a significant waning of anti-XBB.1.16, EG.5.1 and -XBB.1 nAbs was observed both in HI and nHI but nAbs in HI were higher than in nHI. At T6 both HI and nHI individuals displayed low mean levels of anti-XBB.1.16 and EG.5.1 nAbs. Regarding T cell response, IFN-γ values were stable over time and similar in HI and nHI. Our data showed that in PLWH, during the prevalent circulation of the XBB.1.16, EG.5.1, and other XBB sublineages, a bBD mRNA vaccine might not confer broad protection against them. With a view to the 2023/2024 vaccination campaign, the use of the monovalent XBB.1.5 mRNA vaccine should be urgently warranted in PWH to provide adequate protection.

Mpox is a zoonotic disease historically reported in Africa. Since 2003, limited outbreaks have occurred outside Africa. In 2022, the global spread of cases with sustained interhuman transmission and unusual disease features raised public health concerns. We explore the mpox outbreak in Rio de Janeiro (RJ) state, Brazil, in an observational study of mpox-suspected cases from June to December 2022. Data collection relied on a public healthcare notification form. Diagnosis was determined by MPXV-PCR. In 46 confirmed cases, anti-VACV IgG was determined by ELISA, and seven MPXV genomes were sequenced. A total of 3,095 cases were included, 816 (26%) with positive MPXV-PCR results. Most positive cases were men in their 30s and MSM. A total of 285 (35%) MPXV-PCR+ patients lived with HIV. Eight were coinfected with varicella-zoster virus. Anogenital lesions and adenomegaly were associated with the diagnosis of mpox. Females and individuals under 18 represented 9% and 5% of all confirmed cases, respectively, showing higher PCR cycle threshold values and fewer anogenital lesions than adult men. Anti-VACV IgG was detected in 29/46 (63%) patients. All analyzed sequences belonged to clade IIb. In RJ state, mpox presented a diverse clinical picture, represented mainly by mild cases with low complication rates and prominent genital involvement. The incidence in females and children was higher than usually reported. The observation of a bimodal distribution of Ct values, with few positive results, may suggest the need to review the diagnostic criteria in these groups.

Background: While dysfunctional exhausted CD8+ T cells hamper viral control when children acquire hepatitis B virus (HBV) infection, it’s crucial to recognize that CD8+ T cells have diverse phenotypes and functions. This study explored a subset of CD8+ T cells expressing C - C chemokine receptor type 5 (CCR5) in children with HBV infection. Methods: 36 patients in the immune tolerant (IT) group, 33 patients in the immune active (IA) group, and 55 patients in the combined response (CR) group were enrolled. The frequency, functional molecules and effector functions of the CCR5+CD8+ T cells population in different groups were evaluated. Results: The frequency of CCR5+CD8+ T cells correlated positively with the frequency of CCR5+ CD4+ T cells and patient age, and it correlated negatively with ALT, AST, HBV DNA, HBsAg and lactic dehydrogenase levels. CCR5+CD8+ T cells had higher levels of inhibitory and activated receptors and produced higher levels of IFN-γ, IL-2, and TNF-α than CCR5-CD8+ T cells. Conclusion：CCR5+CD8+T cells were partially exhausted but possessed a stronger antiviral activity than CCR5-CD8+T cells. The identification of this subset increases our understanding of CD8+ T cell functions and serve as a potential immunotherapeutic target for children with HBV infection.

Comment on “The risk assessment of uveitis after COVID-19 diagnosis”Patrick Wu,1 Chin-Yuan Yii,2 Su-Boon Yong3,4,5College of Medicine, Lake Erie College of Osteopathic Medicine, Bradenton, FL 34211, USADivision of Gastroenterology and Hepatology, Department of Internal Medicine, Landseed International Hospital, Taoyuan, TaiwanDepartment of Allergy and Immunology, China Medical University Children’s Hospital, Taichung, TaiwanDepartment of Medicine, College of Medicine, China Medical University, Taichung, TaiwanCenter for Allergy, Immunology, and Microbiome (A.I.M.), China Medical University Hospital, Taichung, Taiwan.Corresponding authors:Chin-Yuan Yii, MDDivision of Gastroenterology and Hepatology, Department of Internal Medicine, Landseed International Hospital, Taoyuan, TaiwanAddress: No.77, Guangtai Rd., Pingzhen Dist., Taoyuan City 32449, Taiwan Telephone: +886 3 494 1234Email: [email protected]. Su-Boon Yong, MD PhDDepartment of Medicine, College of Medicine, China Medical University, Taichung, TaiwanAddress: No. 2, Yuh-Der Road, Taichung City 404, TaiwanTelephone: 00886-4-22052121Email: [email protected] author:Patrick Wu, BSCollege of Medicine, Lake Erie College of Osteopathic Medicine, Bradenton, FL 34211, USAAddress: 5000 Lakewood Ranch Blvd, Bradenton, FL 34211, USATelephone: +1(301)3375805Email: [email protected] availability statement: Not applicable. This letter to the editor does not require data collection.Funding statement: Not applicable. Authorship for letter to the editor did not receive any funding.Conflict of interest disclosure: The authors do not have any conflict of interests to declare.Ethics approval statement: Not applicable.Patient consent statement: Not applicable.Permission to reproduce material from other sources: Not applicable.Clinical trial registration: Not applicable.AbstractSeveral suggestions were made for the study by Hsia et al1 regarding uveitis risk following COVID-19 diagnosis. We recommend the authors align the racial composition of study groups more closely with that of U.S. demographics. In addition, we recommend the study to include possibility of false negatives from PCR testing. Lastly, we suggest the authors to consider cases of self-limiting uveitis and relapses independent of COVID-19.Keywords: uveitis risk, COVID-19, racial composition, PCR testing, self-limiting uveitis, relapsesTo the Editor,We read with great interest the study by Hsia et al1regarding the risk assessment of uveitis after COVID-diagnosis. We appreciate the authors’ attention to detail by eliminating possible confounding variables that may contribute to uveitis development. Furthermore, we were impressed by the robust study design incorporating propensity score matching, long follow-ups, and immense study size of more than 4 million patients using the TriNetX analytics platform.Nevertheless, to enhance this study, we recommend the following considerations.First, this study utilized the US research network, covering about 92 million patients to form a COVID-19 cohort and a non-COVID-19 control group, each with 2 million patients, of which 62.5% and 62.4% were white, respectively. This underrepresents the white population by 13% compared to the 75.5% white representation in the 2022 US census. Future studies should align the racial composition of study groups more closely with national demographics.Second, the authors identified COVID-19 cases based on positive PCR tests or antibody immunoassays. Yet, Binny et al’s study in New Zealand illustrates how PCR test sensitivity fluctuates across the COVID-19 infection timeline, influenced by viral load and patient age.2 The sensitivity of PCR tests peaks at 92.7% between 4 to 5 days post-infection, then drops to 88% from 5 to 14 days, while specificity remains near 100%.2 This indicates a higher likelihood of false negatives and very low false positives in PCR testing. Highlighting this significant limitation in the discussion section would be beneficial.Third, this study excluded those diagnosed with uveitis within 6 months before COVID-19 infection, potentially overlooking undiagnosed, self-limiting cases that may resurface post-infection. 10% of intermediate uveitis cases resolve on their own, and anterior uveitis, while often self-limiting, can cause severe complications.3,4 Moreover, uveitis relapses, as reported in Grunwald et al’s study, could occur independently of COVID-19, leading to misattribution of these cases to COVID-19.5 These aspects represent potential limitations of the study that warrant discussion.In conclusion, the study by Hsia et al1 is a major milestone towards incorporating uveitis assessment among COVID-19 patients in healthcare guidelines. This study has tremendous potential to save numerous patients from glaucoma, cataracts, and permanent vision loss. To improve this study, we recommend authors to adjust study groups so that white patients are adequately represented, discuss the possibility of false negatives from PCR testing, and consider cases of self-limiting uveitis and relapses independent of COVID-19.Acknowledgements: noneReferencesHsia NY, Hsu AY, Wang YH, Li JX, Chen HS, Wei JC, Lin CJ, Tsai YY. The risk assessment of uveitis after COVID-19 diagnosis: A multicenter population-based study. J Med Virol. 2023 Oct;95(10):e29188. doi: 10.1002/jmv.29188. PMID: 37881132.Binny RN, Priest P, French NP, Parry M, Lustig A, Hendy SC, Maclaren OJ, Ridings KM, Steyn N, Vattiato G, Plank MJ. Sensitivity of Reverse Transcription Polymerase Chain Reaction Tests for Severe Acute Respiratory Syndrome Coronavirus 2 Through Time. J Infect Dis. 2022 Dec 28;227(1):9-17. doi: 10.1093/infdis/jiac317. PMID: 35876500; PMCID: PMC9384503.Maggon R. INTERMEDIATE UVEITIS PARS PLANITIS. Med J Armed Forces India. 2001 Jan;57(1):84-5. doi: 10.1016/S0377-1237(01)80106-4. Epub 2011 Jul 21. PMID: 27365593; PMCID: PMC4925061.Islam N, Pavesio C. Uveitis (acute anterior). BMJ Clin Evid. 2010 Apr 8;2010:0705. PMID: 21736765; PMCID: PMC2907596.Grunwald L, Newcomb CW, Daniel E, Kaçmaz RO, Jabs DA, Levy-Clarke GA, Nussenblatt RB, Rosenbaum JT, Suhler EB, Thorne JE, Foster CS, Kempen JH; Systemic Immunosuppressive Therapy for Eye Diseases Cohort Study. Risk of relapse in primary acute anterior uveitis. Ophthalmology. 2011 Oct;118(10):1911-5. doi: 10.1016/j.ophtha.2011.02.044. Epub 2011 Jun 16. PMID: 21680024; PMCID: PMC3179829.

Human papillomavirus type 16 (HPV16) is the most common cause of cervical cancer, but most infections are transient and with lesions not progressing to cancer. There is a lack of specific biomarkers for diagnosis and risk stratification. This study aimed to explore the intra-host HPV16 genomic variation in longitudinal samples from HPV16-infected women with different cervical lesion severity (normal, low-grade, and high-grade). The TaME-seq deep sequencing protocol was used to generate whole genome HPV16 sequences of 102 samples collected over time from 40 individuals. Single nucleotide variants (SNVs) and intra-host single nucleotide variants (iSNVs) were identified in the viral genomes. A majority of individuals had a unique set of SNVs and these SNVs were stable over time. Overall, the number of iSNVs and APOBEC3-induced iSNVs were significantly lower in high-grade relative to normal and low-grade samples, respectively. A significant increase in the number of APOBEC3-induced iSNVs over time was observed for normal samples when compared to high-grade. Our results provide new insight into the dynamics of HPV16 within-hosts evolution. Low number of iSNVs and APOBEC3-induced iSNVs, characteristics of high-grade lesions, could potentially serve as biomarkers to guide triage of HPV-induced cervical precancerous lesions.

Infection with influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant risk to human life, health, and the global economy. Vaccination is one of the most effective strategies in the fight against infectious viruses. In this study, we, for the first time, have evaluated the immunogenicity and protective effect of an influenza/SARS-CoV-2 Omicron subunit combined vaccine adjuvanted with MF59 and administered to BALB/c mice. Results showed that the combined vaccine induced high levels of IgG, IgG 1, and IgG 2a antibodies, as well as influenza A H1N1/California/2009 virus-specific hemagglutination-inhibiting antibodies in BALB/c mice. Moreover, this subunit combined vaccine induced high titers of neutralization antibodies against SARS-CoV-2 Omicron BA.5 pseudovirus and effectively reduced the viral load of authentic SARS-CoV-2 Omicron BA.5.2 variant in the cell culture supernatants. These results suggested that this subunit combined vaccine achieved protective effect against both H1N1 A/California/07/2009 strain and SARS-CoV-2 Omicron BA.5.2 variant. It is therefore expected that this study will establish the scientific foundation for the next-step development of combined vaccines against other strains or variants of IAV and SARS-CoV-2.

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.

Title: COVID-19 and Autoimmunity: Do We Need More Evidence?

Background: The clearance or transcriptional silencing of integrated HBV DNA is crucial for achieving a functional cure in patients with chronic hepatitis B (CHB) and reducing the risk of hepatocellular carcinoma (HCC) development. The PLC/PRF/5 cell line is commonly used as an in vitro model for studying HBV integration. In this study, we employed a range of multi-omics techniques to gain a panoramic understanding of the characteristics of HBV integration in PLC/PRF/5 cells and to reveal the transcriptional regulatory mechanisms of integrated HBV DNA.Methods: Transcriptome long-read sequencing (ONT) was conducted to analyze and characterize the transcriptional activity of different HBV DNA integration sites in PLC/PRF/5 cells. Additionally, we collected data related to epigenetic regulation, including whole-genome bisulfite sequencing (WGBS), histone chromatin immunoprecipitation sequencing (ChIP-seq), and assays for transposase-accessible chromatin using sequencing (ATAC-seq), to explore the potential mechanisms involved in the transcriptional regulation of integrated HBV DNA.Result: Long-read RNA sequencing analysis revealed significant transcriptional differences at various integration sites in the PLC/PRF/5 cell line, with higher HBV DNA transcription levels at integration sites on chr11, chr13, and the chr13/chr5 fusion chromosome t (13:5). Combining long-read DNA and RNA sequencing results, we found that transcription of integrated HBV DNA generally starts downstream of the SP1, SP2, or XP promoters. ATAC-seq data confirmed that chromatin accessibility has limited influence on the transcription of integrated HBV DNA in the PLC/PRF/5 cell line. Analysis of whole-genome bisulfite sequencing data showed that the methylation intensity of integrated HBV DNA was highly negatively correlated with its transcription level (r = -0.8929, p = 0.0123). After AzaD treatment, the transcription level of integrated HBV DNA significantly increased, especially for the integration chr17, which had the highest level of methylation. Through ChIP-seq data, we observed the association between histone modification of H3K4me3 and H3K9me3 with the transcription of integrated HBV DNA.

In this study, hesperetin was shown to inhibit the replication of multiple poxviruses, including buffalopox virus (BPXV), vaccinia virus, and lumpy skin disease virus (LSDV). Hesperetin mainly suppressed viral protein synthesis without affecting other steps of the viral life cycle such as attachment, entry, and budding. In a chromatin immunoprecipitation (CHIP) assay, we further demonstrated that hesperetin-induced reduction in BPXV protein synthesis is due to disruption of the binding of the 5’ cap of viral mRNA with the cellular translation initiation factor eIF4E. The molecular docking and MD simulation studies, also confirmed binding of the hesperetin with the cap-binding pocket of eIF4E, in a similar conformation as m7GTP binds. In a BPXV egg infection model, hesperetin was shown to suppress the development of pock lesions on the chorioallantoic membrane, as well as the associated mortality of the chicken embryos. Most importantly, long-term culture of BPXV in the presence of hesperetin did not induce the generation of drug-resistant viral mutants. In conclusion, we for the first time demonstrated the antiviral activity of hesperetin against poxviruses, besides providing novel mechanistic insights into the antiviral action of hesperetin.

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

Respiratory syncytial virus is associated with lower respiratory tract infections. As several types and genotypes can circulate at the same time, genomic characterisation is important for timely epidemiological control and treatment measures. In the last 6 seasons (2017-2023), 191236 nasopharyngeal swabs were processed for respiratory viruses. The incidence of RSV reached 7% in the pre-pandemic season. RSV was most frequent in children under 5 years of age (12.6%), but was also significant in those over 70 years of age (5.63%). The measures taken to control SARS-Cov2 infection were useful for RSV control and the incidence decreased to 1.8%, but caused a change in the types. Pre-pandemic, the majority circulating types were RSV-B/RSV-B/RSV-A and in pandemic it was RSV-B/RSV-B. In the last season, RSV-B and RSV-A were detected in the same proportion. Genetic characterization showed three new clades. This has been taken into account in order to take the correct measures.

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.

Introduction: Acute viral gastroenteritis is a common illness especially in developing countries. Globally, the role of human astroviruses (HAstV) in gastroenteritis among children and elderly is well documented. But there exists a substantial dearth of information on HAstV strains circulating in Nigeria. Method: Viral like particles (VLPs) were purified from stool samples from children diagnosed with acute flaccid paralysis (AFP) between January and December 2020 from five states in Nigeria, using the NetoVIR protocol. Extracted viral RNA and DNA were subjected to a reverse transcription step, and subsequent random PCR amplification. Library preparation and Illumina sequencing were performed. Using the Virome Paired-End Reads (ViPER) pipeline, raw reads were processed into genomic contigs. Phylogenetic and pairwise identity analysis of the recovered HAstV genomes was performed. Results: Six near complete genome sequences of HAstV were identified and classified as HAstV 4 (n=1), HAstV5 (n=1), HAstV8 (n=1) and MLB-3 (n=3). The HAstV5 belonged to a yet unclassified sub-lineage which we tentatively named HAstV-5d. Phylogenetic analysis of ORFs 1a, 1b and 2 suggested recombination events inside the MAstV1 species. Furthermore, phylogenetic analysis implied a geographic linkage between the HAstV5 strain from this study with two strains from Cameroon across all the genomic regions. Conclusion: We report for the first time the circulation of HAstV genotypes 4, 8 and MLB-3 in Nigeria and present data suggestive for the existence of a new sub-lineage of HAstV5. To further understand the burden, diversity, and evolution of HAstV increased research interest as well as robust HAstV surveillance in Nigeria is essential.

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.