Impact of ADAR-induced editing of minor viral RNA populations on replication and transmission of SARS-CoV-2.

Adenosine deaminases acting on RNA (ADAR) are RNA-editing enzymes that may restrict viral infection. We have utilized deep sequencing to determine adenosine to guanine (A→G) mutations, signifying ADAR activity, in clinical samples retrieved from 93 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients in the early phase of the COVID-19 pandemic. A→G mutations were detected in 0.035% (median) of RNA residues and were predominantly nonsynonymous. These mutations were rarely detected in the major viral population but were abundant in minor viral populations in which A→G was more prevalent than any other mutation (P < 0.001). The A→G substitutions accumulated in the spike protein gene at positions corresponding to amino acids 505 to 510 in the receptor binding motif and at amino acids 650 to 655. The frequency of A→G mutations in minor viral populations was significantly associated with low viral load (P < 0.001). We additionally analyzed A→G mutations in 288,247 SARS-CoV-2 major (consensus) sequences representing the dominant viral population. The A→G mutations observed in minor viral populations in the initial patient cohort were increasingly detected in European consensus sequences between March and June 2020 (P < 0.001) followed by a decline of these mutations in autumn and early winter (P < 0.001). We propose that ADAR-induced deamination of RNA is a significant source of mutated SARS-CoV-2 and hypothesize that the degree of RNA deamination may determine or reflect viral fitness and infectivity.

Brush Samples of Oral Lesions to FTA Elute Card for High-risk Human Papilloma Virus Diagnosis.

AIM: To investigate the level of agreement between three non-invasive methods for hrHPV diagnosis in oral and oropharyngeal squamous cell carcinoma (OSCC, OPSCC) and in oral mucosal lesions. MATERIALS AND METHODS: For hrHPV DNA FTA Elute card™ and Anyplex II HPV28™ were used and for hrHPV mRNA PreTect SEE™ in tumour patients (n=60), non-tumour lesions (n=51), immunosuppression or previous hrHPV-infection (n=32). RESULTS: The level of agreement between the DNA-methods was 82.2% (k=0.54, p=0.001). Pair-wise comparison for the FTA Elute card were close to the reference (AUC=0.83, 95% CI=0.73-0.90). hrHPV mRNA was diagnosed in 50% of the tumours, with an agreement level of 58.3%, compared to Anyplex II (k=0.17, p=0.04). The hrHPV positivity in oral lesions was 3.9% for immunosuppression and for previous HPV infection 9.4%. CONCLUSION: The FTA card is reliable for hrHPV DNA diagnosis while mRNA gives an insight into viral activity and correlates with severity of the lesion.

High-risk human papillomavirus in patients with oral leukoplakia and oral squamous cell carcinoma-A multi-centre study in Sweden, Brazil and Romania.

OBJECTIVES: Although causal associations between oral leukoplakia (OL), oral squamous cell carcinoma (OSCC) and high-risk human papillomavirus (HR-HPV) have been speculated upon in several reports, conclusive evidence has not been presented. This study investigates whether the number of cases of HR-HPV in OL has increased over time and whether the prevalence of HR-HPV-positive OL differs in various parts of the world. PATIENTS AND METHODS: A total of 432 patients with OL from Sweden, Brazil and Romania were analysed. Patients were divided into historical (1992-2002) and contemporary (2011-2017) cohorts from the respective countries. Seventeen patients with OL developed oral squamous cell carcinoma (OSCC). A real-time PCR assay, targeting HPV sub-types 6,11,16,18,31,33,35,39,45,52,56,58 and 59, was performed to detect HR-HPV in patients with OL. RESULTS: In the Swedish and Romanian cohorts, none of the investigated HPV sub-types were detected. In the Brazilian cohorts, five patients with OL (3%) were positive for HR-HPV, including four patients from the contemporary cohort (HPV 16, 31, 33) and one from the historical cohort (HPV 11). All the cases of OL that transformed into OSCC were HR-HPV-negative, as were the corresponding tumours. CONCLUSIONS: In summary, the prevalence of HR-HPV in OL is low in all the tested countries, and the incidence has not changed over time. HR-HPV in OL does not seem to be a driver of oncogenesis.

Humoral immunity to tetanus, diphtheria and polio in adults after treatment for hematological malignancies.

INTRODUCTION: After chemotherapy, children with acute lymphocytic leukemia lose immunity and need revaccination against tetanus and diphtheria. However, little is known about immunity in adult patients after treatment for hematological malignancies. In this study, we assessed serology levels against polio, diphtheria and tetanus in adult patients after conventional treatment for leukemia and lymphoma. PATIENTS: One hundred and four patients, age 61 (19-86) years, were included at a median of 18 (4-77) months after chemotherapy for acute leukemia (n = 24) or lymphoma (n = 80). Pre-treatment sera were available in 73 cases for a pre-versus post treatment comparison. Healthy, age- and sex matched controls were available for 47 pts. METHODS: Tetanus antibodies were quantified using ELISA, and antibody levels ≥0.01 IU/mL were considered protective. Diphtheria antibodies were analyzed using neutralization test (n = 60) or by ELISA (n = 44). In both tests values ≥0.01 IU/mL were considered protective. Antibodies against poliovirus serotype 1 and 3 were assessed by a neutralizing test. A microneutralization titer of ≥2 was considered protective. RESULTS: Tetanus: There were significantly more non-immune patients after treatment (24%), compared to before (12%), p = 0.02. Post-treatment antibody levels were significantly lower than pre-treatment levels (p = 0.02). Diphtheria: There was a trend, p = 0.06, towards more non-immune patients after treatment (21%) compared to before (27%). Antibody levels post treatment were lower than pre treatment levels (p = 0.03) and lower than controls (p = 0.01). Polio: There was no significant difference in the number of non-immune patients before vs after chemotherapy for either PV1 or PV3. Protective immunity against serotype 1 and 3 was preserved in 90 and 97%, respectively. CONCLUSIONS: After standard chemotherapy for leukemia and lymphoma a significant proportion of patients had impaired humoral immunity to diphtheria and tetanus. However, polio immunity was well preserved.

Combined Testing of p16 Tumour-suppressor Protein and Human Papillomavirus in Patients With Oral Leukoplakia and Oral Squamous Cell Carcinoma.

BACKGROUND: Oral leukoplakia (OL) is a potentially malignant oral mucosal disorder. A casual association between OL, oral squamous cell carcinoma (OSCC) and human papillomavirus (HPV) infection has been suggested, but no conclusive evidence has been presented. p16, a tumour-suppressor protein, is used as a surrogate marker for HPV infection. The aim of this study was to investigate how overexpression of p16 correlates with HPV infection in OL and in OSCC. PATIENTS AND METHODS: Seventy-four patients with OL and 13 with OSCC with p16 overexpressed, were analyzed by immunohistochemistry visualizing p16 and a real-time polymerase chain reaction (PCR) assay targeting HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 52, 56, 58 and 59. RESULTS: Overexpression of p16 was observed in 18% of patients with OL. None of the HPV subtypes were detected by PCR analysis in patients with OL. In the p16-positive OSCC specimens, 38% were also HPV16-positive. CONCLUSION: Overexpression of p16 was not found to be a reliable biomarker for HPV infection in patients with OL and OSCC.

Type-dependent E6/E7 mRNA expression of single and multiple high-risk human papillomavirus infections in cervical neoplasia.

BACKGROUND: Coinfection with multiple HPV types is common in cervical lesions, but the biological significance of the individual infections is difficult to establish. Expression of oncogenic E6/E7 HPV mRNA is correlated to risk of malignant progression, commercial assays for genotyping E6/E7 mRNA of all HR-HPV are lacking. OBJECTIVES: To characterize the tendency of 12 HR-HPV to express mRNA, correlated to the severity of the cervical lesion. Furthermore, we wanted to analyse mRNA expression in multiple infections, in order to establish which genotype may be responsible for cellular transformation. STUDY DESIGN: 245 samples from women with normal histology, various grades of dysplasia (cervical intraepithelial neoplasia grade 1-3), and cancer, were analysed for presence and genotyping of HPV DNA and mRNA using an in house real-time PCR test. RESULTS: Presence of mRNA was detected for 64% of the in total 422 HPV infections present in the samples, and more commonly in high-grade lesions. In 88% of DNA-positive samples from CIN2+ lesions, mRNA could be detected, compared to 33% of DNA-positive samples from women in screening with normal cytology. The genotype most prone to express mRNA in high-grade lesions was HPV45, followed by HPV16 and HPV31, less prone was HPV59. Expression of mRNA was significantly enhanced in CIN2+ lesions, an association also found for HPV16. In 52% of multiple infections (in which mRNA expression was generally more common), more than one genotype expressed mRNA, a phenomenon increasing with severity of lesion. Presence of mRNA could more often be detected in samples with multiple infections than in samples with single infections. CONCLUSIONS: The frequent expression of E6/E7 by HPV45 may promote oncogenicity and could be of clinical importance. Since presence of E6/E7 mRNA was common in multiple infections regardless of histology, multiple infection could be a clinically important finding. In multiple HPV infections, mRNA testing may identify the genotype that causes transformation. However, since mRNA expression of several genotypes in one sample is common, further and larger studies using complementary techniques are required.

Type-specific human papillomavirus E6/E7 mRNA detection by real-time PCR improves identification of cervical neoplasia.

DNA-based human papillomavirus (HPV) assays show high sensitivity but poor specificity in detecting high-grade cervical lesions. Assays detecting mRNA of the oncoproteins E6 and E7 show higher specificity but lack either detection of all high-risk HPV genotypes or the capacity to specify the detected genotypes. Therefore, a real-time PCR assay detecting type-specific E6/E7 mRNA was developed and the clinical performance evaluated. A total of 210 cervical LBC (liquid-based cytology) samples from 204 women were analyzed for HPV DNA and mRNA with the in-house real-time PCR as well as PreTect HPV-Proofer. The sensitivity of real-time PCR mRNA detection to identify histologically confirmed CIN2+ (cervical intraepithelial neoplasia, grade 2 or higher) was 0.91, compared to 0.95 for DNA analysis. The specificity was 0.68 compared to 0.38, and the positive predictive value (PPV) was higher for mRNA (0.67 versus 0.52) without any loss in negative predictive value (NPV). The sensitivity of the real-time PCR mRNA test was somewhat higher than that for PreTect HPV-Proofer (0.83 versus 0.75) in analyses for the same genotypes. The specificities were similar (0.76 versus 0.77). In analyses for mRNA of the eight most common genotypes in cervical cancer (HPV16, -18, -31, -33, -35, -45, -52, and -58), the sensitivity of detection of CIN2+ lesions was 0.87 and the specificity 0.74, with a PPV of 0.70. In conclusion, real-time PCR for detection of HPV E6/E7 mRNA transcripts can be a sensitive and specific tool in screening and investigation of cervical neoplasia. The composition of HPV types in mRNA testing needs to be further investigated to optimize sensitivity and specificity.

Activity of the small modified amino acid alpha-hydroxy glycineamide on in vitro and in vivo human immunodeficiency virus type 1 capsid assembly and infectivity.

Upon maturation of the human immunodeficiency virus type 1 (HIV-1) virion, proteolytic cleavage of the Gag precursor protein by the viral protease is followed by morphological changes of the capsid protein p24, which will ultimately transform the virus core from an immature spherical to a mature conical structure. Virion infectivity is critically dependent on the optimal semistability of the capsid cone structure. We have reported earlier that glycineamide (G-NH(2)), when added to the culture medium of infected cells, inhibits HIV-1 replication and that HIV-1 particles with aberrant core structures were formed. Here we show that it is not G-NH(2) itself but a metabolite thereof, alpha-hydroxy-glycineamide (alpha-HGA), that is responsible for the antiviral activity. We show that alpha-HGA inhibits the replication of clinical HIV-1 isolates with acquired resistance to reverse transcriptase and protease inhibitors but has no effect on the replication of any of 10 different RNA and DNA viruses. alpha-HGA affected the ability of the HIV-1 capsid protein to assemble into tubular or core structures in vitro and in vivo, probably by binding to the hinge region between the N- and C-terminal domains of the HIV-1 capsid protein as indicated by matrix-assisted laser desorption ionization-mass spectrometry results. As an antiviral compound, alpha-HGA has an unusually simple structure, a pronounced antiviral specificity, and a novel mechanism of antiviral action. As such, it might prove to be a lead compound for a new class of anti-HIV substances.

Tracing of norovirus outbreak strains in mussels collected near sewage effluents.

Noroviruses from mussels collected near sewage effluents were compared with local patient outbreak strains. Sequence analyses of RNA polymerase-capsid-poly(A)-3' (3.1-kilobase) regions confirmed the 99.9% similarity between genotype I.1 strains from mussels and patient strains from recreational-bathing outbreaks, indicating the potential usefulness of sentinel norovirus mussel studies in tracing human norovirus contamination of coastal waters.

Real-time Taqman PCR targeting 14 human papilloma virus types.

BACKGROUND: Subtyping of human papilloma virus (HPV) may enhance the precision of vaginal cytological assessments and will be important for investigating the effect of the recently introduced vaccine against types 16 and 18. OBJECTIVES AND STUDY DESIGN: To evaluate an in-house real-time PCR targeting HPV types 16-18-31-33-35-39-45-51-52-56-58-59-6-11, by analysing 107 liquid-based cytology specimens representing various degrees of dysplasia. RESULTS: In all, 71 samples were HPV positive, with multiple types present in 37 (52%). Comparison with Roche Linear Array on a subset of 24 of these 71 samples showed a good agreement. One or several types were detected in 17/17 (100%) samples with cervical intraepithelial neoplasia grade 2-3 (CIN 2-3), 16/19 (84%) with CIN 1, 32/43 (74%) with Atypical Squamous Cells of Undetermined Significance (ASCUS), and in 6/28 (21%) with benign cytology. Estimates of mean viral load were lower in CIN 1-3 than in ASCUS ( approximately 4000 vs. approximately 25,000 copies/1000 cells), and clearly lower in samples with benign cytology ( approximately 50 copies/1000 cells). CONCLUSION: The HPV rates in groups with different degrees of dysplasia agrees with previous reports and support a strong link between types 16/18 and severe dysplasia. The high rate of multiple type infection might influence the outcome of HPV vaccination. The possible importance of viral load should be further studied.

Outbreak of norovirus in Västra Götaland associated with recreational activities at two lakes during August 2004.

A large community outbreak of norovirus (NV) gastrointestinal infection occurred in Västra Götaland County, Sweden in August 2004, following attendance at recreational lakes. A frequency age-matched case control study was undertaken of persons who had attended these lakes to identify risk factors. 163 cases and 329 controls were included. Analysis indicates that having water in the mouth while swimming (OR=4.7; 95% CI 1.1-20.2), attendance at the main swimming area at Delsjön Lake (OR=25.5; 95% CI 2.5-263.8), taking water home from a fresh water spring near Delsjön lake (OR=17.3; 95% CI 2.7-110.7) and swimming less than 20 m from shore (OR=13.4; 95% CI 2.0-90.2) were significant risk factors. The probable vehicle was local contamination of the lake water (especially at the main swimming area). The source of contamination could not be determined.

Predictive factors and virological response to interferon treatment in children with chronic hepatitis B.

Further knowledge about factors predicting response to interferon treatment for chronic hepatitis B in children is required, in particular as the benefits of therapy are uncertain. In the present study, baseline characteristics were related to virological and histological responses in 27 children given interferon-alpha for 24 weeks after steroid priming. HBe seroconversion was seen in 8 of 27 HBeAg positive patients and was accompanied by a sustained virological response (SR), with a median 4.1 log HBV DNA reduction. Pretreatment viraemia level was the only baseline parameter associated with SR. After 12 weeks of IFN (mid-treatment), viraemia was significantly reduced in all patients, with a median of 3.0 (range 0.6-5.2) log decline in SR compared with 0.6 (range -0.5-3.6) log decline in non-sustained responders (NSR). HBV DNA levels below 1 million copies/ml at week 12 predicted sustained response with a positive predictive value of 75% and a negative predictive value of 89%. During the latter half of the IFN treatment HBV DNA tended to increase by a mean of 0.4-0.5 log for all patient groups. Flares during IFN treatment were rare or mild as measured by ALT. Pretreatment anti-HBc IgM was associated with liver damage but not with response. Histological inflammation scores were improved in SR. Thus, pretreatment HBV DNA levels were associated with IFN response, and the virological response at week 12 predicts SR and may be useful in the decision to continue or modify therapy.

Glycine-amide is an active metabolite of the antiretroviral tripeptide glycyl-prolyl-glycine-amide.

The chemically modified tripeptide glycyl-prolyl-glycine-amide (GPG-NH(2)) inhibits replication of human immunodeficiency virus (HIV) type 1 (HIV-1) in vitro, probably by interfering with capsid formation. The aim of the present study was to determine whether the metabolites glycyl-proline (GP-OH), glycine (G-OH), prolyl-glycine-amide (PG-NH(2)), proline (P-OH), and glycine-amide (G-NH(2)) from proteolytic cleavage may inhibit the replication of HIV-1 in vitro. PG-NH(2) has previously been shown to have a modest effect on HIV-1 replication. In the present study we show that G-NH(2) exhibits a pronounced inhibitory effect on HIV-1. This effect was not due to a decrease in cell proliferation or viability and could not be shown for herpes simplex virus type 1. The G-NH(2) concentration that inhibited virus replication by 50% (IC(50)) was equimolar to that of GPG-NH(2) and ranged from 3 to 41 microM. Transmission electron microscopy revealed that the effect of G-NH(2) on HIV-1 morphology was equivalent to that of GPG-NH(2) and showed disarranged capsid structures, indicating interference with capsid formation. Serial passage of HIV-infected cells with G-NH(2) for more than 20 subcultivations did not decrease the susceptibility to the compound. The results from this study suggest that GPG-NH(2) might act as a prodrug and that G-NH(2) is an active antiretroviral metabolite.

Obligatory involvement of CD26/dipeptidyl peptidase IV in the activation of the antiretroviral tripeptide glycylprolylglycinamide (GPG-NH(2)).

GPG-NH2 and G-NH2 are highly selective antiretroviral agents in cell culture, and both compounds inhibit HIV replication in CEM cell cultures to an equal extent (50% effective concentration: approximately 30 microM). The lymphocyte surface glycoprotein marker CD26, which is identical to dipeptidyl peptidase IV, efficiently converted GPG-NH2 to G-NH2 releasing the dipeptide GP-OH. The closely related QPG-NH2 derivative was also inhibitory to HIV, presumably by the dipeptidyl peptidase IV (DPP IV)-catalyzed release of G-NH2. In contrast, the cyclic pQPG-NH2 derivative in which the glutamine at the amino terminal position of QPG-NH2 was replaced by pyroglutamine and which is resistant to cleavage by purified CD26, was devoid of antiviral activity. CD26 is abundantly expressed on a variety of HIV target cells and is also present in serum of bovine, murine and human origin. The CD26/DPP IV enzymatic activity in serum and in cell suspensions could be efficiently inhibited by the CD26/DPP IV inhibitor L-isoleucinepyrrolidine (IlePyr) with 50% inhibitory concentrations ranging between 20 and 100 microM. When combined in HIV-1-infected cell cultures, IlePyr and Diprotin A (DP-A), another CD26/DPP IV inhibitor, abrogated the antiviral activity of GPG-NH2 but not of G-NH2. Therefore, it was concluded that the anti-HIV drug GPG-NH2 is not active as such, but rather behaves as a prodrug that must be obligatorily cleaved by CD26/DPP IV to G-NH2 to exert its antiretroviral activity. This is the first demonstration of a lymphocyte activation/differentiation marker (i.e. CD26) that plays a direct regulatory and indispensable role in the eventual antiretroviral activity of small synthetic molecules such as the antiretroviral (pro)drug GPG-NH2.

No cross-resistance or selection of HIV-1 resistant mutants in vitro to the antiretroviral tripeptide glycyl-prolyl-glycine-amide.

The chemically modified tripeptide glycyl-prolyl-glycine-amide (GPG-NH(2)) inhibits replication of HIV-1 in vitro, probably by interfering with capsid formation. This study was aimed at determining cross-resistance between antiretroviral drugs and GPG-NH(2), and whether resistance to GPG-NH(2) can be induced in vitro. Fifty-five clinical HIV-1 isolates with different resistance-related mutations were tested for susceptibility to GPG-NH(2). No correlation between NRTI-, NNRTI- or PI-resistance and efficacy of GPG-NH(2) was found, indicating the lack of cross-resistance. Serial passages were performed with GPG-NH(2), and with lamivudine, and genotypic or phenotypic changes were determined. Resistance to lamivudine was detected after six passages. No resistance to GPG-NH(2) was generated after 30 passages in two parallel series. However, one mutation (T107I) in the p24 gene was detected in both series, but this mutation was not associated with decreased sensitivity to GPG-NH(2).

Histologic activity of childhood chronic hepatitis B related to viremia levels, genotypes, mutations, and epidemiologic factors.

BACKGROUND: Despite high viral load, children with chronic hepatitis B virus (HBV) infection may lack significant biochemical signs of liver dysfunction. Failure to develop abnormal liver chemistriesis is probably due to immunologic hyporeactivity. Despite the absence of biochemical abnormalities in these patients, there is still a risk for long-term complications. The pathogenic importance of viral load and genetic variability is less well studied in children than in adults. METHODS: We evaluated viremia levels, genotypes, and mutations related to histologic evidence of liver damage in 71 HBV carriers, aged 2 to 18 years, all of non-Swedish origin. RESULTS: None of the of 22 children who were hepatitis B e antigen (HBeAg) negative had severe liver disease or had HBV DNA levels greater than 10 copies/mL (mean 10 ); 3 (14%) of them had increased alanine aminotransferase (ALT). The 49 HBeAg-positive children had a mean HBV DNA level of 10 copies/mL, and increased ALT was seen in 28 (55%). Core promoter mutations (at nt 1764) or precore mutations (at codon 1, 2, or 28) were rare; they were seen in four and one HBeAg-positive children, and in four and nine HBeAg-negative children, respectively, without association to liver damage. C-1858 was associated with more liver inflammation. Genotype did not significantly influence liver damage. Children with horizontal transmission had a faster rate of seroconversion and more inflammation of the liver. CONCLUSIONS: Severe HBeAg-negative hepatitis with high HBV DNA levels and mutations in the core promoter or precore regions seems to be less common in children than in adults. C-1858 strains may be more pathogenic, but this requires further study. Epidemiologic factors influence the course of infection.

Genotype mixtures of hepatitis B virus in patients treated with interferon.

Little is known about coinfection among several hepatitis B virus (HBV) genotypes, although previous reports of recombination and genotype shifts indicate that this should occur. In the present study, we designed a method to identify mixtures of genotype A and another genotype, regardless of whether one of them predominates. Using this method, signs of genotypic coinfection were found in 20 (67%) of 30 hepatitis B e antigen-positive patients treated with interferon (IFN). In 8 of these patients, coinfection or genotype shifts were detectable by direct sequencing or standard preS genotyping. In most of these cases, genotype changes were detected after a >2-log decrease or increase of the HBV DNA level. The presence of genotype mixtures did not significantly influence IFN response. Because quasi-species selection may occur at or shortly after transmission, patients might acquire HBV infection from subjects who appear to be infected with a different genotype. This should be considered when tracing the source of HBV infection.

Mutations in the X region and core promoter are rare and have little impact on response to interferon therapy for chronic hepatitis B.

Therapy for chronic hepatitis B with interferon-alpha (IFN) may result in viral clearance and hepatitis B e seroconversion in 30-40% of patients. It is still unclear whether viral genetic variability influences response rates. However, certain core promoter mutations were recently associated with a better response to IFN. In the present study, the entire X region, including the core promoter, of hepatitis B virus (HBV) from 26 HBeAg-positive patients treated with IFN for 12 weeks, was sequenced. Serum samples pre-treatment, at end-of-treatment, and at follow-up of 18 sustained and 8 nonsustained responders were analyzed. Most patients were of European origin and had moderate aminotransferase elevation (mean 2.4 x upper limit of normal) and genotype A infection. Before treatment, 16 patients had an X region identical to a consensus sequence of the corresponding genotype; in the remaining 10 patients, a median of 1.5 mutations were found. After treatment, 1-4 new mutations (mean 1.8) had emerged in 5 patients. There was no association between specific mutations, or the number of mutations, and response to IFN. The low frequency of mutations indicates that analysis of this region is of limited clinical value and that emerging mutations in this region are not major determinants of response to treatment with IFN-alpha.