Production of infectious reporter murine norovirus by VP2 trans-complementation.

Human norovirus (HuNoV) causes gastroenteritis, a disease with no effective therapy or vaccine, and does not grow well in culture. Murine norovirus (MNV) easily replicates in cell cultures and small animals and has often been used as a model to elucidate the structural and functional characteristics of HuNoV. An MNV plasmid-based reverse genetics system was developed to produce the modified recombinant virus. In this study, we attempted to construct the recombinant virus by integrating a foreign gene into MNV ORF3, which encodes the minor structural protein VP2. Deletion of VP2 expression abolished infectious particles from MNV cDNA clones, and supplying exogenous VP2 to the cells rescued the infectivity of cDNA clones without VP2 expression. In addition, the coding sequence of C-terminal ORF3 was essential for cDNA clones compensated with VP2 to produce infectious particles. Furthermore, the recombinant virus with exogenous reporter genes in place of the dispensable region of ORF3 was propagated when VP2 was constitutively supplied. Our findings indicate that foreign genes can be transduced into the norovirus ORF3 region when VP2 is supplied and that successive propagation of modified recombinant norovirus could lead to the development of norovirus-based vaccines or therapeutics.IMPORTANCEIn this study, we revealed that some of the coding regions of ORF3 could be replaced by a foreign gene and infectious virus could be produced when VP2 was supplied. Propagation of this virus depended on VP2 being supplied in trans, indicating that this virus could infect only once. Our findings help to elucidate the functions of VP2 in the virus lifecycle and to develop other caliciviral vectors for recombinant attenuated live enteric virus vaccines or therapeutics tools.

Human transmission and outbreaks of feline-like G6 rotavirus revealed with whole-genome analysis of G6P[9] feline rotavirus.

Group A rotaviruses (RVAs) are generally highly species-specific; however, some strains infect across species. Feline RVAs sporadically infect humans, causing gastroenteritis. In 2012 and 2013, rectal swab samples were collected from 61 asymptomatic shelter cats at a public health center in Mie Prefecture, Japan, to investigate the presence of RVA and any association with human infections. The analysis identified G6P[9] strains in three cats and G3P[9] strains in two cats, although no feline RVA sequence data were available for the former. A whole-genome analysis of these G6P[9] strains identified the genotype constellation G6-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3. The nucleotide identity among these G6P[9] strains exceeded 99.5% across all 11 gene segments, indicating the circulation of this G6P[9] strain among cats. Notably, strain RVA/Human-wt/JPN/KF17/2010/G6P[9], previously detected in a 3-year-old child with gastroenteritis, shares high nucleotide identity (>98%) with Mie20120017f, the representative G6P[9] strain in this study, across all 11 gene segments, confirming feline RVA infection and symptomatic presentation in this child. The VP7 gene of strain Mie20120017f also shares high nucleotide identity with other sporadically reported G6 RVA strains in humans. This suggests that feline-origin G6 strains as the probable source of these sporadic G6 RVA strains causing gastroenteritis in humans globally. Moreover, a feline-like human G6P[8] strain circulating in Brazil in 2022 was identified, emphasizing the importance of ongoing surveillance to monitor potential global human outbreaks of RVA.

Different immunological responses following immunization with two mRNA vaccines.

INTRODUCTION: Immunological responses were investigated following immunization with two mRNA vaccines: BNT162b2 and mRNA-1273. METHODS: Neutralizing antibody (NAb) was assayed before, 2-4 weeks after, and 3 and 6 months after the primary immunization, and the same time-points after booster dose with 6- or 8-months interval. Whole-blood culture was stimulated with spike antigen, and cytokine production was assayed. RESULTS: NAb was detected after primary immunization, NAb titers began to decrease three months after primary immunization with BNT162b2, lower than those after mRNA-1273, and elevated after booster immunization. The NAb level was 1/2 lower against δ variant, and 1/16 lower against omicron variant in comparison with that against α variant. Cytokine production following immunization with mRNA-1273 was maintained within three months at higher levels of Th1 (TNF-α), Th2 (IL-4 and IL-5), and inflammatory cytokines (IL-6 and IL-17) than that following immunization with BNT162b2, reflecting prominent levels of NAb following immunization with mRNA-1273. Cytokine production decreased six months after primary immunization in both vaccine recipients and was enhanced following booster doses. During the omicron outbreak, medical staff members in the outpatient office experienced asymptomatic infection, with a greater than 4-fold increase in NAb titers against omicron variant even after booster immunization. Asymptomatic infection enhanced the production of Th2 and inflammatory cytokines. CONCLUSION: mRNA-1273 induced stronger NAb responses with wide-range cross-reactive antibodies against δ and omicron variants. mRNA-1273 induced higher levels of Th1, Th2, and inflammatory cytokines than BNT162b2 did, reflecting higher levels of NAb against variant strains.

Luciferase-based quantification of membrane fusion induced by SARS-CoV-2 S protein.

The ongoing COVID-19 pandemic is caused by SARS-CoV-2. Although several effective vaccines that target the Spike protein on the viral surface have been deployed, additional therapeutic agents are urgently needed. Here, we developed a system to measure the Spike protein function by quantifying cellular membrane fusion induced by the Spike protein. The system enables the evaluation of the effects of drugs and neutralizing antibodies against SARS-CoV-2 without using live viruses. Furthermore, the system characterizes membrane fusion activity of the Spike protein of each variant to reveal that Delta variant has more potent than Wuhan and Omicron. Our system could lead to develop high-throughput screening for drug candidates and neutralization antibodies that target virus entry and characterize Spike proteins from variants.

Nasal delivery of single-domain antibody improves symptoms of SARS-CoV-2 infection in an animal model.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed, particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients.

Dynamic rotation of the protruding domain enhances the infectivity of norovirus.

Norovirus is the major cause of epidemic nonbacterial gastroenteritis worldwide. Lack of structural information on infection and replication mechanisms hampers the development of effective vaccines and remedies. Here, using cryo-electron microscopy, we show that the capsid structure of murine noroviruses changes in response to aqueous conditions. By twisting the flexible hinge connecting two domains, the protruding (P) domain reversibly rises off the shell (S) domain in solutions of higher pH, but rests on the S domain in solutions of lower pH. Metal ions help to stabilize the resting conformation in this process. Furthermore, in the resting conformation, the cellular receptor CD300lf is readily accessible, and thus infection efficiency is significantly enhanced. Two similar P domain conformations were also found simultaneously in the human norovirus GII.3 capsid, although the mechanism of the conformational change is not yet clear. These results provide new insights into the mechanisms of non-enveloped norovirus transmission that invades host cells, replicates, and sometimes escapes the hosts immune system, through dramatic environmental changes in the gastrointestinal tract.

Histo-blood group antigens of glycosphingolipids predict susceptibility of human intestinal enteroids to norovirus infection.

The molecular mechanisms behind infection and propagation of human restricted pathogens such as human norovirus (HuNoV) have defied interrogation because they were previously unculturable. However, human intestinal enteroids (HIEs) have emerged to offer unique ex vivo models for targeted studies of intestinal biology, including inflammatory and infectious diseases. Carbohydrate-dependent histo-blood group antigens (HBGAs) are known to be critical for clinical infection. To explore whether HBGAs of glycosphingolipids contribute to HuNoV infection, we obtained HIE cultures established from stem cells isolated from jejunal biopsies of six individuals with different ABO, Lewis, and secretor genotypes. We analyzed their glycerolipid and sphingolipid compositions and quantified interaction kinetics and the affinity of HuNoV virus-like particles (VLPs) to lipid vesicles produced from the individual HIE-lipid extracts. All HIEs had a similar lipid and glycerolipid composition. Sphingolipids included HBGA-related type 1 chain glycosphingolipids (GSLs), with HBGA epitopes corresponding to the geno- and phenotypes of the different HIEs. As revealed by single-particle interaction studies of Sydney GII.4 VLPs with glycosphingolipid-containing HIE membranes, both binding kinetics and affinities explain the patterns of susceptibility toward GII.4 infection for individual HIEs. This is the first time norovirus VLPs have been shown to interact specifically with secretor gene-dependent GSLs embedded in lipid membranes of HIEs that propagate GII.4 HuNoV ex vivo, highlighting the potential of HIEs for advanced future studies of intestinal glycobiology and host-pathogen interactions.

Impact of the Heterogeneity in Free Chlorine, UV254, and Ozone Susceptibilities Among Coxsackievirus B5 on the Prediction of the Overall Inactivation Efficiency.

The disinfection susceptibilities of viruses vary even among variants, yet the inactivation efficiency of a certain virus genotype, species, or genus was determined based on the susceptibility of its laboratory strain. The objectives were to evaluate the variability in susceptibilities to free chlorine, UV254, and ozone among 13 variants of coxsackievirus B5 (CVB5) and develop the model allowing for predicting the overall inactivation of heterogeneous CVB5. Our results showed that the susceptibilities differed by up to 3.4-fold, 1.3-fold, and 1.8-fold in free chlorine, UV254, and ozone, respectively. CVB5 in genogroup B exhibited significantly lower susceptibility to free chlorine and ozone than genogroup A, where the laboratory strain, Faulkner, belongs. The capsid protein in genogroup B contained a lower number of sulfur-containing amino acids, readily reactive to oxidants. We reformulated the Chick-Watson model by incorporating the probability distributions of inactivation rate constants to capture the heterogeneity. This expanded Chick-Watson model indicated that up to 4.2-fold larger free chlorine CT is required to achieve 6-log inactivation of CVB5 than the prediction by the Faulkner strain. Therefore, it is recommended to incorporate the variation in disinfection susceptibilities for predicting the overall inactivation of a certain type of viruses.

Functional receptor molecules CD300lf and CD300ld within the CD300 family enable murine noroviruses to infect cells.

Norovirus is the leading cause of acute gastroenteritis worldwide. Since the discovery of human norovirus (HuNoV), an efficient and reproducible norovirus replication system has not been established in cultured cells. Although limited amounts of virus particles can be produced when the HuNoV genome is directly transfected into cells, the HuNoV cycle of infection has not been successfully reproduced in any currently available cell-culture system. Those results imply that the identification of a functional cell-surface receptor for norovirus might be the key to establishing a norovirus culture system. Using a genome-wide CRISPR/Cas9 guide RNA library, we identified murine CD300lf and CD300ld as functional receptors for murine norovirus (MNV). The treatment of susceptible cells with polyclonal antibody against CD300lf significantly reduced the production of viral progeny. Additionally, ectopic CD300lf expression in nonsusceptible cell lines derived from other animal species enabled MNV infection and progeny production, suggesting that CD300lf has potential for dictating MNV host tropism. Furthermore, CD300ld, which has an amino acid sequence in the N-terminal region of its extracellular domain that is highly homologous to that of CD300lf, also functions as a receptor for MNV. Our results indicate that direct interaction of MNV with two cell-surface molecules, CD300lf and CD300ld, dictates permissive noroviral infection.

Evaluation of the use of various rat strains for immunogenic potency tests of Sabin-derived inactivated polio vaccines.

Slc:Wistar rats have been the only strain used in Japan for purpose of evaluating a national reference vaccine for the Sabin-derived inactivated polio vaccine (sIPV) and the immunogenicity of sIPV-containing products. However, following the discovery that the Slc:Wistar strain was genetically related to the Fischer 344 strain, other "real" Wistar strains, such as Crlj:WI, that are available worldwide were tested in terms of their usefulness in evaluating the immunogenicity of the past and current lots of a national reference vaccine. The response of the Crlj:WI rats against the serotype 1 of sIPV was comparable to that of the Slc:Wistar rats, while the Crlj:WI rats exhibited a higher level of response against the serotypes 2 and 3. The immunogenic potency units of a national reference vaccine determined using the Slc:Wistar rats were reproduced on tests using the Crlj:WI rats. These results indicate that a titer of the neutralizing antibody obtained in response to a given dose of sIPV cannot be directly compared between these two rat strains, but that, more importantly, the potency units are almost equivalent for the two rat strains.

First complete genome sequences of genogroup V, genotype 3 porcine sapoviruses: common 5'-terminal genomic feature of sapoviruses.

Sapoviruses (SaVs) are enteric viruses and have been detected in various mammals. They are divided into multiple genogroups and genotypes based on the entire major capsid protein (VP1) encoding region sequences. In this study, we determined the first complete genome sequences of two genogroup V, genotype 3 (GV.3) SaV strains detected from swine fecal samples, in combination with Illumina MiSeq sequencing of the libraries prepared from viral RNA and PCR products. The lengths of the viral genome (7494 nucleotides [nt] excluding polyA tail) and short 5'-untranslated region (14 nt) as well as two predicted open reading frames are similar to those of other SaVs. The amino acid differences between the two porcine SaVs are most frequent in the central region of the VP1-encoding region. A stem-loop structure which was predicted in the first 41 nt of the 5'-terminal region of GV.3 SaVs and the other available complete genome sequences of SaVs may have a critical role in viral genome replication. Our study provides complete genome sequences of rarely reported GV.3 SaV strains and highlights the common 5'-terminal genomic feature of SaVs detected from different mammalian species.

Complete genome analysis of porcine kobuviruses from the feces of pigs in Japan.

Porcine kobuviruses (PoKoVs) are ubiquitously distributed in pig populations worldwide and are thought to be enteric viruses in swine. Although PoKoVs have been detected in pigs in Japan, no complete genome data for Japanese PoKoVs are available. In the present study, 24 nearly complete or complete sequences of the PoKoV genome obtained from 10 diarrheic feces and 14 non-diarrheic feces of Japanese pigs were analyzed using a metagenomics approach. Japanese PoKoVs shared 85.2-100% identity with the complete coding nucleotide (nt) sequences and the closest relationship of 85.1-98.3% with PoKoVs from other countries. Twenty of 24 Japanese PoKoVs carried a deletion of 90 nt in the 2B coding region. Phylogenetic tree analyses revealed that PoKoVs were not grouped according to their geographical region of origin and the phylogenetic trees of the L, P1, P2, and P3 genetic regions showed topologies different from each other. Similarity plot analysis using strains from a single farm revealed partially different similarity patterns among strains from identical farm origins, suggesting that recombination events had occurred. These results indicate that various PoKoV strains are prevalent and not restricted geographically on pig farms worldwide and the coexistence of multiple strains leads to recombination events of PoKoVs and contributes to the genetic diversity and evolution of PoKoVs.

Identification of further diversity among posaviruses.

Recently, there have been reports of new members of posavirus-like viruses in the order Picornavirales. In this study, using a metagenomics approach, 11 posavirus-like sequences (>7,000 nucleotides) were detected in 155 porcine fecal samples. Phylogenetic analysis revealed that the newly identified virus sequences, together with other posavirus-like viruses, form distinct clusters within the order Picornavirales, composed of eight genogroups and unassigned sequences based on amino acid sequences of the helicase and RNA-dependent RNA polymerase regions, with <40 % and <50 % sequence identity, respectively. We propose further classifications of highly diverse posavirus populations based on newly identified sequences from Japanese pig feces.

Construction and characterization of an infectious cDNA clone of rat hepatitis E virus.

Rat hepatitis E virus (HEV) is related to human HEV and has been detected in wild rats worldwide. Here, the complete genome of rat HEV strain R63/DEU/2009 was cloned downstream of the T7 RNA polymerase promoter and capped genomic RNA generated by in vitro transcription was injected into nude rats. Rat HEV RNA could be detected in serum and faeces of rats injected intrahepatically, but not in those injected intravenously. Rat HEV RNA-positive faecal suspension was intravenously inoculated into nude rats and Wistar rats leading to rat HEV RNA detection in serum and faeces of nude rats, and to seroconversion in Wistar rats. In addition, rat HEV was isolated in PLC/PRF/5 cells from the rat HEV RNA-positive faecal suspension of nude rats and then passaged. The cell culture supernatant was infectious for nude rats. Genome analysis identified nine point mutations of the cell-culture-passaged virus in comparison with the originally cloned rat HEV genome. The results indicated that infectious rat HEV could be generated from the cDNA clone. As rats are widely used and well-characterized laboratory animals, studies on genetically engineered rat HEV may provide novel insights into organ tropism, replication and excretion kinetics as well as immunological changes induced by hepeviruses.

Emergence of Intergenogroup Reassortant G9P[4] Strains Following Rotavirus Vaccine Introduction in Ghana.

Rotavirus (RVA) is a leading cause of childhood gastroenteritis. RVA vaccines have reduced the global disease burden; however, the emergence of intergenogroup reassortant strains is a growing concern. During surveillance in Ghana, we observed the emergence of G9P[4] RVA strains in the fourth year after RVA vaccine introduction. To investigate whether Ghanaian G9P[4] strains also exhibited the DS-1-like backbone, as seen in reassortant G1/G3/G8/G9 strains found in other countries in recent years, this study determined the whole genome sequences of fifteen G9P[4] and two G2P[4] RVA strains detected during 2015-2016. The results reveal that the Ghanaian G9P[4] strains exhibited a double-reassortant genotype, with G9-VP7 and E6-NSP4 genes on a DS-1-like backbone (G9-P[4]-I2-R2-C2-M2-A2-N2-T2-E6-H2). Although they shared a common ancestor with G9P[4] DS-1-like strains from other countries, further intra-reassortment events were observed among the original G9P[4] and co-circulating strains in Ghana. In the post-vaccine era, there were significant changes in the distribution of RVA genotype constellations, with unique strains emerging, indicating an impact beyond natural cyclical fluctuations. However, reassortant strains may exhibit instability and have a limited duration of appearance. Current vaccines have shown efficacy against DS-1-like strains; however, ongoing surveillance in fully vaccinated children is crucial for addressing concerns about long-term effectiveness.

Distribution of Human Sapovirus Strain Genotypes over the Last Four Decades in Japan: a Global Perspective.

Sapovirus (SaV) infections are a public health problem because they cause acute gastroenteritis in humans of all ages, both sporadically and as outbreaks. However, only a limited amount of SaV sequence information, especially whole-genome sequences for all the SaV genotypes, is publicly available. Therefore, in this study, we determined the full/near-full-length genomic sequences of 138 SaVs from the 2001 to 2015 seasons in 13 prefectures across Japan. The genogroup GI was predominant (67%, n = 92), followed by genogroups GII (18%, n = 25), GIV (9%, n = 12), and GV (6%, n = 9). Within the GI genogroup, four different genotypes were identified: GI.1 (n = 44), GI.2 (n = 40), GI.3 (n = 7), and GI.5 (n = 1). We then compared these Japanese SaV sequences with 3,119 publicly available human SaV sequences collected from 49 countries over the last 46 years. The results indicated that GI.1, and GI.2 have been the predominant genotypes in Japan, as well as in other countries, over at least four decades. The 138 newly determined Japanese SaV sequences together with the currently available SaV sequences, could facilitate a better understanding of the evolutionary patterns of SaV genotypes.

A critical role of MYC for transformation of human cells by HPV16 E6E7 and oncogenic HRAS.

Human papillomaviruses (HPVs) are the primary causal agents for development of cervical cancer, and deregulated expression of two viral oncogenes E6 and E7 is considered to contribute to disease initiation. Recently, we have demonstrated that transduction of oncogenic HRAS (HRAS(G12V)) and MYC together with HPV16 E6E7 is sufficient for tumorigenic transformation of normal human cervical keratinocytes (HCKs). Here, we show that transduction of HRAS(G12V) on the background of E6E7 expression causes accumulation of MYC protein and tumorigenic transformation of not only normal HCKs but also other normal primary human cells, including tongue keratinocytes and bronchial epithelial cells as well as hTERT-immortalized foreskin fibroblasts. Subcutaneous transplantation of as few as 200 HCKs expressing E6E7 and HRAS(G12V) resulted in tumor formation within 2 months. Dissecting RAS signaling pathways, constitutively active forms of AKT1 or MEK1 did not result in tumor formation with E6E7, but tumorigenic transformation was induced with addition of MYC. Increased MYC expression endowed resistance to calcium- and serum-induced terminal differentiation and activated the mammalian target of rapamycin (mTOR) pathway. An mTOR inhibitor (Rapamycin) and MYC inhibition a level not affecting proliferation in culture both markedly suppressed tumor formation by HCKs expressing E6E7 and HRAS(G12V). These results suggest that a single mutation of HRAS could be oncogenic in the background of deregulated expression of E6E7 and MYC plays a critical role in cooperation with the RAS signaling pathways in tumorigenesis. Thus inhibition of MYC and/or the downstream mTOR pathway could be a therapeutic strategy not only for the MYC-altered but also RAS-activated cancers.

Generation of CRISPR-Cas9-mediated genetic knockout human intestinal tissue-derived enteroid lines by lentivirus transduction and single-cell cloning.

Human intestinal tissue-derived enteroids (HIEs; also called organoids) are a powerful ex vivo model for gastrointestinal research. Genetic modification of these nontransformed cultures allows new insights into gene function and biological processes involved in intestinal diseases as well as gastrointestinal and donor segment-specific function. Here we provide a detailed technical pipeline and protocol for using the CRISPR-Cas9 genome editing system to knock out a gene of interest specifically in HIEs by lentiviral transduction and single-cell cloning. This protocol differs from a previously published alternative using electroporation of human colonoids to deliver piggyback transposons or CRISPR-Cas9 constructs, as this protocol uses a modified, fused LentiCRISPRv2-small-guiding RNA to express Cas9 and small-guiding RNA in a lentivirus. The protocol also includes the steps of gene delivery and subsequent single-cell cloning of the knockout cells as well as verification of clones and sequence identification of the mutation sites to establish knockout clones. An overview flowchart, step-by-step guidelines and troubleshooting suggestions are provided to aid the researcher in obtaining the genetic knockout HIE line within 2-3 months. In this protocol, we further describe how to use HIEs as an ex vivo model to assess host restriction factors for viral replication (using human norovirus replication as an example) by knocking out host attachment factors or innate immunity genes. Other applications are discussed to broaden the utility of this system, for example, to generate knockin or conditional knockout HIE lines to investigate the function of essential genes in many biological processes including other types of organoids.

Experimental Adaptation of Murine Norovirus to Calcium Hydroxide.

Slaked lime (calcium hydroxide) is a commonly used disinfectant for fecal sludge. Although viruses are inactivated by lime treatment, whether RNA viruses adapt to lime treatment has not yet been determined. Here, we show that murine norovirus developed higher tolerance during serial passages with lime treatment. We compared synonymous and non-synonymous nucleotide diversities of the three open reading frames of viral genome and revealed that virus populations were subjected to enhanced purifying selection over the course of serial passages with lime treatment. Virus adaptation to lime treatment was coincident with amino acid substitution of lysine to arginine at position 345 (K345R) on the major capsid protein VP1, which accounted for more than 90% of the population. The infectious clones with the K345R produced using a plasmid-based reverse genetics system exhibited greater tolerance in a lime solution, which indicated that the specific amino acid substitution was solely involved in the viral tolerance in lime treatment.

Genotype-dependent kinetics of enterovirus inactivation by free chlorine and ultraviolet (UV) irradiation.

Inactivation kinetics of enterovirus by disinfection is often studied using a single laboratory strain of a given genotype. Environmental variants of enterovirus are genetically distinct from the corresponding laboratory strain, yet it is poorly understood how these genetic differences affect inactivation. Here we evaluated the inactivation kinetics of nine coxsackievirus B3 (CVB3), ten coxsackievirus B4 (CVB4), and two echovirus 11 (E11) variants by free chlorine and ultraviolet irradiation (UV). The inactivation kinetics by free chlorine were genotype- (i.e., susceptibility: CVB5 < CVB3 ≈ CVB4 < E11) and genogroup-dependent and exhibited up to 15-fold difference among the tested viruses. In contrast, only minor (up to 1.3-fold) differences were observed in the UV inactivation kinetics. The differences in variability between the two disinfectants could be rationalized by their respective inactivation mechanisms: inactivation by UV mainly depends on the genomic size and composition, which was similar for all viruses tested, whereas free chlorine targets the viral capsid protein, which exhibited critical differences between genogroups and genotypes. Finally, we integrated the observed variability in inactivation rate constants into an expanded Chick-Watson model to estimate the overall inactivation of an enterovirus consortium. The results highlight that the distribution of inactivation rate constants and the abundance of each genotype are essential parameters to accurately predict the overall inactivation of an enterovirus population by free chlorine. We conclude that predictions based on inactivation data of a single variant or reference pathogen alone likely overestimate the true disinfection efficiency of free chlorine.