Characterization of virus‒host recombinant variants of the hepatitis E virus.

Hepatitis E virus is a single-strand, positive-sense RNA virus that can lead to chronic infection in immunocompromised patients. Virus-host recombinant variants (VHRVs) have been described in such patients. These variants integrate part of human genes into the polyproline-rich region that could introduce new post-translational modifications (PTMs), such as ubiquitination. The aim of this study was to characterize the replication capacity of different VHRVs, namely, RNF19A, ZNF787, KIF1B, EEF1A1, RNA18, RPS17, and RPL6. We used a plasmid encoding the Kernow strain, in which the fragment encoding the S17 insertion was deleted (Kernow p6 delS17) or replaced by fragments encoding the different insertions. The HEV RNA concentrations in the supernatants and the HepG2/C3A cell lysates were determined via RT-qPCR. The capsid protein ORF2 was immunostained. The effect of ribavirin was also assessed. The HEV RNA concentrations in the supernatants and the cell lysates were higher for the variants harboring the RNF19A, ZNF787, KIF1B, RPS17, and EEF1A1 insertions than for the Kernow p6 del S17, while it was not with RNA18 or RPL6 fragments. The number of ORF2 foci was higher for RNF19A, ZNF787, KIF1B, and RPS17 than for Kernow p6 del S17. VHRVs with replicative advantages were less sensitive to the antiviral effect of ribavirin. No difference in PTMs was found between VHRVs with a replicative advantage and those without. In conclusion, our study showed that insertions did not systematically confer a replicative advantage in vitro. Further studies are needed to determine the mechanisms underlying the differences in replicative capacity. IMPORTANCE: Hepatitis E virus (HEV) is a major cause of viral hepatitis. HEV can lead to chronic infection in immunocompromised patients. Ribavirin treatment is currently used to treat such chronic infections. Recently, seven virus-host recombinant viruses were characterized in immunocompromised patients. These viruses have incorporated a portion of a human gene fragment into their genome. We studied the consequences of these insertions on the replication capacity. We found that these inserted fragments could enhance virus replication for five of the seven recombinant variants. We also showed that the recombinant variants with replicative advantages were less sensitive to ribavirin in vitro. Finally, we found that the mechanisms leading to such a replicative advantage do not seem to rely on the post-translational modifications introduced by the human gene fragment that could have modified the function of the viral protein. The mechanisms involved in improving the replication of such recombinant viruses remain to be explored.

HPV genotyping in clinical samples using long-read single-molecule real-time sequencing.

Human papillomavirus (HPV) genotyping is widely used, particularly in combination with high-risk (HR) HPV tests for cervical cancer screening. We developed a genotyping method using sequences of approximately 800 bp in the E6/E7 region obtained by PacBio single molecule real-time sequencing (SMRT) and evaluated its performance against MY09-11 L1 sequencing and after the APTIMA HPV genotyping assay. The levels of concordance of PacBio E6/E7 SMRT sequencing with MY09-11 L1 sequencing and APTIMA HPV genotyping were 100% and 90.8%, respectively. The sensitivity of PacBio E6/EA7 SMRT was slightly greater than that of L1 sequencing and, as expected, lower than that of HR-HPV tests. In the context of cervical cancer screening, PacBio E6/E7 SMRT is then best used after a positive HPV test. PacBio E6/E7 SMRT genotyping is an attractive alternative for HR and LR-HPV genotyping of clinical samples. PacBio SMRT sequencing provides unbiased genotyping and can detect multiple HPV infections and haplotypes within a genotype.

BK Viremia and Viruria Does Not Depend on the Type of Double-J Stent Used During Kidney Transplantation.

OBJECTIVES: BK virus is a major cause of chronic renal allograft failure.Transplant ureteral stent use has been reported as a risk factorfor BK virus infection. Recently, the use of a new type of ureteral stent (Magnetic Black Star) was reported in kidney transplant recipients. The aim ofthis preliminary report was to compare BK virus viremia and viruria occurrence depending on the type of double-J stent (standard versus Magnetic Black Star). MATERIALS AND METHODS: We included all kidney transplants performed in our center from January to December 2022. Each case had double-J stent placement. Indwelling stents were either a 6- or 7-Fr standard double-J stent or a 6-Fr Magnetic Black Star double-J stent. The type of double-J stent was chosen according to the surgeon's preference. A standard BK virus screening protocol was followed during the study period, which consisted of routine polymerase chain reaction examination of plasma and urine samples during monthly follow-ups. RESULTS: We assessed 120 patients without missing data: 92 patients received standard double-J stents and 28 patients received Magnetic Black Star stents. Patients were mostly male in the standard group (70.7%) versus the Magnetic Black Star group (42.9%) (P = .01). ABO- and HLA-incompatible transplant rates were similar in both groups. BK viremia occurrence and BK viruria occurrence were similar between groups at 1 month, 3 months, and 6 months. CONCLUSIONS: This preliminary study showed no differences concerning BKvirus infection depending on the type of double-J stents used during kidney transplant.

Increased Prevalence of Antibodies to Hepatitis E Virus in Patients with Neurocysticercosis.

We explored the association between serological status for hepatitis E and neurocysticercosis (NCC) in neurologic patients attending a national neurological referral center in Lima, Perú, between the years 2008 and 2012. Anti-hepatitis E antibodies were evaluated in patients with and without NCC, and a control group of rural general population. Anti-hepatitis E IgG was found in 23.8% of patients with NCC, compared with 14.3% in subjects without NCC from a general rural population (P = 0.023) and 14.4% in subjects with neurological complaints without NCC (P = 0.027). Seropositive patients had a median age of 44 years compared with 30 years in seronegative patients (P <0.001). No significant differences in sex, region of residence, or liver enzyme values were found. Seropositivity to hepatitis E was frequent in this Peruvian population and higher in patients with NCC, suggesting shared common routes of infection.

Study of the cellular and humoral immune responses to SARS-CoV-2 vaccination.

Our understanding of cellular immunity in response to COVID-19 infection or vaccination is limited because of less commonly used techniques. We investigated both the cellular and humoral immune responses before and after the administration of a third dose of the SARS-CoV-2 vaccine among a group of healthcare workers. Cellular immunity was evaluated using the VIDAS interferon-gamma (IFNγ) RUO test, which enables automated measurement of IFNγ levels after stimulating peripheral blood lymphocytes. Booster doses significantly enhanced both cellular and humoral immunity. Concerning cellular response, the booster dose increased the percentage of positive IFNγ release assay (IGRA) results but no difference in IFNγ release was found. The cellular response was not associated with protection against SARS-CoV-2 infection. Interestingly, vaccinated and infected healthcare workers exhibited the highest levels of anti-spike and neutralizing antibodies. In conclusion, the IGRA is a simple method for measuring cellular immune responses after vaccination. However, its usefulness as a complement to the study of humoral responses is yet to be demonstrated in future research.

Evaluation of an automated platform for the detection of HEV RNA in plasma and stool.

INTRODUCTION: We evaluated the performance of the automated Altostar HEV RNA platform for detecting HEV RNA. METHODS AND RESULTS: Clinical performance was determined by testing 81 plasma samples and 10 fecal samples manually quantified previously with the Realstar RT-PCR assay using the Magnapure instrument for extraction. The assays were concordant for 79/81 plasma samples (97.5%) and 10/10 (100%) fecal samples. The two plasma samples that tested negative with the Altostar assay had a very low HEV RNA concentration (1.6 and 1.4 log10 IU/ml). Quantitative results obtained with the automated platform and the manual workflow were highly correlated (ρ= 0.98, p<0.01). The intra-run and inter-run standard deviation were 0.09 IU/ml and 0.13 IU/ml respectively. The assay was linear from 2 to 6 log IU/ml. The limit of detection determined by Probit analysis with the WHO HEV RNA standard was 7.6 [95% CI: 4.4-52.5] IU/ml. CONCLUSIONS: The Altostar platform enables highly accurate testing for the detection of HEV RNA in stool and the quantification of HEV RNA in plasma. This allowed us to shorten turnaround times and to save time for the technical staff.

Effect of steroid pulses in severe BK virus allograft nephropathy with extensive interstitial inflammation.

INTRODUCTION: As there is no specific antiviral treatment currently available for BK polyomavirus associated nephropathy (BKVAN), its management relies on immunosuppression reduction in kidney transplant patients. Data on efficacy of steroid pulses in this indication are lacking. METHODS: We performed a retrospective monocenter study on 64 patients diagnosed with biopsy-proven BKVAN. Patients within the "pulse group" (n = 37) received IV methylprednisolone 10 mg/kg 3 days consecutively. In the "low dose" steroid group (n = 27), patients were continued oral prednisone 5 mg daily. RESULTS: Mean follow up was 78 months in the steroid pulse group and 56 months in the low dose group (p = 0.15). Mean eGFR values at diagnosis were comparable, as well as other demographic characteristics. Mean BK plasma viral load was higher in "pulse" than in "low dose" steroid group. Pulse group had higher inflammation and tubulitis (p < 0.05). Graft loss reached 57% in the "pulse" group versus 41% in the "low dose" group, p = 0.20. Rejection events were similar. No major adverse event was statistically associated with steroid pulse, including infections, cancer, and de novo diabetes. CONCLUSION: No significant differences were found in the evolution of both groups of patients, despite patients receiving "pulse" steroids were identified as the most severe sharing higher BK viral load and more frequent active lesions on histology.

Intact proviruses are enriched in the colon and associated with PD-1+TIGIT- mucosal CD4+ T cells of people with HIV-1 on antiretroviral therapy.

BACKGROUND: The persistence of intact replication-competent HIV-1 proviruses is responsible for the virological rebound off treatment. The gut could be a major reservoir of HIV-1 due to the high number of infected target cells. METHODS: We collected blood samples and intestinal biopsies (duodenum, ileum, colon) from 42 people with HIV-1 receiving effective antiretroviral therapy. We used the Intact Proviral DNA Assay to estimate the frequency of intact HIV-1 proviruses in the blood and in the intestinal mucosa of these individuals. We analyzed the genetic complexity of the HIV-1 reservoir by performing single-molecule next-generation sequencing of HIV-1 env DNA. The activation/exhaustion profile of mucosal T lymphocytes was assessed by flow cytometry. FINDINGS: Intact proviruses are particularly enriched in the colon. Residual HIV-1 transcription in the gut is associated with persistent mucosal and systemic immune activation. The HIV-1 intestinal reservoir appears to be shaped by the proliferation of provirus-hosting cells. The genetic complexity of the viral reservoir in the colon is positively associated with TIGIT expression but negatively with PD-1, and inversely related to its intact content. The size of the intact reservoir in the colon is associated with PD-1+TIGIT- mucosal CD4+ T cells, particularly in CD27+ memory cells, whose proliferation and survival could contribute to the enrichment of the viral reservoir by intact proviruses. INTERPRETATION: Enrichment in intact proviruses makes the gut a key compartment for HIV-1 persistence on antiretroviral therapy. FUNDING: This project was supported by grants from the ANRS-MIE (ANRS EP61 GALT), Sidaction, and the Institut Universitaire de France.

Human papillomavirus testing using HPV APTIMA® assay and an external cellularity control in self-collected samples.

In cervical cancer screening programs, the detection of high-risk human papillomavirus (HR-HPV) is now widely implemented on physician-collected samples and has expanded to include self-collected samples. The use of a cellularity control (CC) is needed to reduce false-negative HPV results. An external mRNA CC for the HPV APTIMA® assay was assessed for its analytical performance and the results were compared with both cervix cytobrush samples taken by physicians and self-collected vaginal samples from 148 women. The performance of the CC was adjusted to control for the presence of cellular mRNA in the ThinPrep® and Multitest® transport media. This CC is user-friendly but implies to perform two independent assays on PANTHER® automate. Self-collected vaginal sampling gives a lower median CC results (13.2 vs. 16.9 min) but a higher risk of negative CC results (3.3 vs. 0%). The usefulness of the CC for the HR-HPV assay may be optimized by the definition of a threshold for a minimum cell number to be tested to increase confidence in HPV-negative results. The systematic use of an RNA CC increases confidence for HPV RNA assays on self-collected vaginal samples.

Evaluation of two anti-SARS-CoV-2 antibody immunoassays for monitoring patients on pre-exposure prophylaxis.

Pre-exposure prophylaxis (PrEP) is crucial to prevent severe COVID-19 in immunocompromised patients. A reliable method is needed to quantify anti-SARS-CoV-2 antibody levels for personalized monitoring during PrEP. We measured the binding antibody concentrations of 63 immunocompromised patients receiving 300mg or 600mg tixagevimab/cilgavimab on PrEP day and twice during the following 3 months. All blood samples were tested using the Abbott anti-SARS-CoV-2 IgG II Quant assay, the Roche Elecsys anti-SARS-CoV-2 S assay, and live virus-based neutralization assays. The results of the two immunoassays were correlated on day 0, 1 month, and 3 months post-PrEP. Passing-Bablok regression demonstrated higher anti-S concentration values measured with the Roche immunoassay compared to those measured with the Abbott immunoassay. Antibody concentrations were higher after 600 mg tixagevimab/cilgavimab prophylaxis than after 300 mg. The neutralizing antibody titers obtained using the omicron BA.5 and BA.2.75 strains were low. Both automated immunoassays are suitable for monitoring immunocompromised patients on PrEP.

SARS-CoV-2 Co-Infections and Recombinations Identified by Long-Read Single-Molecule Real-Time Sequencing.

Co-infection with at least 2 strains of virus is the prerequisite for recombination, one of the means of genetic diversification. Little is known about the prevalence of these events in SARS-CoV-2, partly because it is difficult to detect them. We used long-read PacBio single-molecule real-time (SMRT) sequencing technology to sequence whole genomes and targeted regions for haplotyping. We identified 17 co-infections with SARS-CoV-2 strains belonging to different clades in 6829 samples sequenced between January and October, 2022 (prevalence 0.25%). There were 3 Delta/Omicron co-infections and 14 Omicron/Omicron co-infections (4 cases of 21K/21L, 1 case of 21L/22A, 2 cases of 21L/22B, 4 cases of 22A/22B, 2 cases of 22B/22C and 1 case of 22B/22E). Four of these patients (24%) also harbored recombinant minor haplotypes, including one with a recombinant virus that was selected in the viral quasispecies over the course of his chronic infection. While co-infections remain rare among SARS-CoV-2-infected individuals, long-read SMRT sequencing is a useful tool for detecting them as well as recombinant events, providing the basis for assessing their clinical impact, and a precise indicator of epidemic evolution. IMPORTANCE SARS-CoV-2 variants have been responsible for the successive waves of infection over the 3 years of pandemic. While co-infection followed by recombination is one driver of virus evolution, there have been few reports of co-infections, mainly between Delta and Omicron variants or between the first 2 Omicron variants 21K_BA.1 and 21L_BA.2. The 17 co-infections we detected during 2022 included cases with the recent clades of Omicron 22A, 22B, 22C, and 22E; 24% harbored recombinant variants. This study shows that long-read SMRT sequencing is well suited to SARS-CoV-2 genomic surveillance.

Weaker Effects of the Fourth Dose of BNT162b2 SARS-CoV-2 Vaccine on the Elderly Human Population.

The vaccines presently available are less effective in older people due to senescence of their immune systems. We measured the antibody responses of 42 adults living in nursing homes after the third and the fourth doses of an mRNA vaccine and found that the strain (BA.2 and BA.2.75: from 64 to 128, BA.5: from 16 to 32, BQ.1.1: from 16 to 64 among the uninfected) influenced the effect of the fourth dose of vaccine on neutralizing antibodies. The fourth dose also increased binding antibodies (from 1036 BAU/mL to 5371 BAU/mL among the uninfected, from 3700 BAU/mL to 6773 BAU/mL among the BA.5 infected). This effect was less significant than that of the third dose of vaccine for both neutralizing (BA.2: from 8 to 128, BA.5: from 2 to 16, BA.2.75: from 8 to 64, BQ.1.1: from 2 to 16) and binding antibodies (from 139.8 BAU/mL to 2293 BAU/mL). However, the fourth dose attained the 5000 BAU/mL threshold conferring approximately 80% protection against a SARS-CoV-2 BA.2 infection in most individuals, unlike the third.

Clustered Cases of Waterborne Hepatitis E Virus Infection, France.

The identification of seven cases of hepatitis E virus infection in a French rural hamlet in April 2015 led to investigations confirming the clustering and identifying the source of the infection. Laboratories and general practitioners in the area actively searched for other cases based on RT-PCR and serological tests. The environment, including water sources, was also checked for HEV RNA. Phylogenetic analyses were performed to compare HEV sequences. No other cases were found. Six of the seven patients lived in the same hamlet, and the seventh used to visit his family who lived there. All HEV strains were very similar and belonged to the HEV3f subgenotype, confirming the clustering of these cases. All the patients drank water from the public network. A break in the water supply to the hamlet was identified at the time the infection probably occurred; HEV RNA was also detected in a private water source that was connected to the public water network. The water flowing from the taps was quite turbid during the break. The private water supply containing HEV RNA was the likely source of the contamination. Private water supplies not disconnected from the public network are still frequent in rural areas, where they may contribute to public water pollution.

Immunohistologic Features of Cerebral Venous Thrombosis Due to Vaccine-Induced Immune Thrombotic Thrombocytopenia.

OBJECTIVES: Vaccine-induced immune thrombotic thrombocytopenia (VITT), a recently described entity characterized by thrombosis at unusual locations such as cerebral venous sinus and splanchnic vein, has been rarely described after adenoviral-encoded COVID-19 vaccines. In this study, we report the immunohistological correlates in 3 fatal cases of cerebral venous thrombosis related to VITT analyzed at an academic medical center. METHODS: Detailed neuropathologic studies were performed in 3 cases of cerebral venous thrombosis related to VITT after adenoviral COVID-19 vaccination. RESULTS: Autopsy revealed extensive cerebral vein thrombosis in all 3 cases. Polarized thrombi were observed with a high density of neutrophils in the core and a low density in the tail. Endothelial cells adjacent to the thrombus were largely destroyed. Markers of neutrophil extracellular trap and complement activation were present at the border and within the cerebral vein thrombi. SARS-CoV-2 spike protein was detected within the thrombus and in the adjacent vessel wall. DISCUSSION: Data indicate that neutrophils and complement activation associated with antispike immunity triggered by the vaccine is probably involved in the disease process.

Glucocorticoids selectively affect the memory T cell response to SARS-Cov2 spike in vaccinated and post-infected patients with systemic lupus erythematosus.

Immune response to vaccines and pathogens remains unclear in patients with systemic lupus erythematosus (SLE). To investigate this, a single-center retrospective study was conducted with 47 SLE patients vaccinated against COVID-19, including 13 who subsequently developed an asymptomatic/mild disease. As compared to controls, post-vaccine response against Spike was reduced in SLE patients when considering both memory T-cells in a whole blood interferon gamma release assay (IGRA-S) and IgG anti-Spike antibody (Ab) responses. The SLE-associated defective IGRA-S response was associated with a serum albumin level below 40 g/L and with the use of glucocorticoids, while a defective IgG anti-Spike Ab response was associated with lower levels of anti-dsDNA and anti-SSA/Ro 52 kDa Abs. IGRA-S and IgG anti-Spike responses were independent from SLE activity and clinical phenotype, low complement, hypergammaglobulinemia, and lymphopenia. As compared to controls, SLE patients showed a rapid decay of anti-Spike T-cell memory and stable IgG anti-Spike Ab responses. In conclusion, both T cell and humoral anti-Spike responses were independently affected in our SLE patients cohort, which supports the exploration of both responses in the follow-up of SLE patients and especially in those receiving glucocorticoids.

The pressing need for a global HEV vaccine.

Based on the worldwide distribution of hepatitis E virus (HEV) and its ability to cause major epidemics in low-income countries, the global availability of a HEV vaccine is a pressing clinical need. Populations at risk of severe forms of the infection are well characterised: patients with chronic liver disease - at risk of liver failure; pregnant women - at risk of fulminant hepatitis or obstetrical complications; and immunosuppressed patients, particularly those with solid organ transplants - at risk of chronic hepatitis and rapid progression to cirrhosis. Only one hepatitis E vaccine is presently being manufactured. It has been proven to be effective and safe. However, its accessibility, as well as data on its long-term efficacy and the duration of protection it confers, are limited. While individuals considered to be at risk of severe infection appear to be ideal targets for the vaccine, its effectiveness and tolerability have not yet been studied in populations with chronic liver disease and immunosuppressed patients. Hepatitis E vaccination could also play an important role in controlling outbreaks in large waterborne epidemics. Clinical trials on these populations are needed.

Whole-genome single molecule real-time sequencing of SARS-CoV-2 Omicron.

New variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome can only be identified using accurate sequencing methods. Single molecule real-time (SMRT) sequencing has been used to characterize Alpha and Delta variants, but not Omicron variants harboring numerous mutations in the SARS-CoV-2 genome. This study assesses the performance of a target capture SMRT sequencing protocol for whole genome sequencing (WGS) of SARS-CoV-2 Omicron variants and compared it to that of an amplicon SMRT sequencing protocol optimized for Omicron variants. The failure rate of the target capture protocol (6%) was lower than that of the amplicon protocol (34%, p < 0.001) on our data set, and the median genome coverage with the target capture protocol (98.6% [interquartile range (IQR): 86-99.4]) was greater than that with the amplicon protocol (76.6% [IQR: 66-89.6], [p < 0.001]). The percentages of samples with >95% whole genome coverage were 64% with the target capture protocol and 19% with the amplicon protocol (p < 0.05). The clades of 96 samples determined with both protocols were 93% concordant and the lineages of 59 samples were 100% concordant. Thus, target capture SMRT sequencing appears to be an efficient method for WGS, genotyping and detecting mutations of SARS-CoV-2 Omicron variants.