Search Results (69)

Persistent SARS-CoV-2 infections involve prolonged viral replication and immune system interactions, potentially driving viral evolution and immune escape. This study examines viral characteristics and host gene expression changes in persistent infections. The nasopharyngeal samples from four patients with persistent SARS-CoV-2 infections at Tottori University Hospital, Japan, were analyzed. Viral isolates were cultured, and infectivity was assessed using TCID₅₀ assays. To investigate host responses, RNA sequencing (RNA-seq) was performed to identify differentially expressed genes (DEGs), and Gene Ontology (GO) enrichment analysis mapped affected biological pathways. Viral genome sequencing detected mutations associated with prolonged infection. The results showed significant infectivity differences between early- and late-phase infection. Gene expression analysis revealed a strong early phase of pro-inflammatory response (IL6, TNF, IL1B, CXCL10) followed by immune suppression. GO enrichment analysis highlighted inflammation and cytokine-mediated immune pathways. Genomic sequencing identified mutations in ORF1ab and the spike (S) protein, potentially aiding immune escape. The findings underscore that SARS-CoV-2 adapts during persistent infections, altering infectivity and immune responses. These highlight the need for continued monitoring of prolonged infections to mitigate immune escape and viral evolution. Full article

In the past 5 years, the COVID-19 pandemic has experienced frequently changing variants contextualizing immune evasion. The emergence of Omicron with >30–50 mutations on the spike gene has shown a sharp divergence from its relative VOCs, such as WT, Alpha, Beta, Gamma, and Delta. The requisition of prime boosting was essential within 3–6 months to improve the Nab response that had been not lasted for longer. Omicron subvariant BA.1.1 was less transmissible, but with an extra nine mutations in next variant BA.2 made it more transmissible. This remarkable heterogeneity was reported in ORF1ab or TRS sites, ORF7a, and 10 regions in the genomic sequences of Omicron BA.2 and its evolving subvariants BA.4.6, BF.7, BQ.2, BF. 7, BA.2.75.2, and BA.5 (BQ.1 and BQ.1.1). The mutational stability of subvariants XBB, XBB 1, XBB 1.5, and XBB 1.6 conferred a similar affinity towards ACE-2. This phenomenon has been reported in breakthrough infections and after booster vaccinations producing hybrid immunity. The reduced pathogenic nature of Omicron has implicated its adaptation either through immunocompromised individuals or other animal hosts. The binding capacity of RBD and ACE-2, including the proteolytic priming via TMPRSS2, reveals its (in-vitro) transmissibility behavior. RBD mutations signify transmissibility, S1/S2 enhances virulence, while S2 infers the effective immunogenic response. Initial mutations D614G, E484A, N501Y, Q493K, K417N, S477N, Y505H, and G496S were found to increase the Ab escape. Some mutations such as, R346K, L452R, and F486Vwere seen delivering immune pressure. HR2 region (S2) displayed mutations R436S, K444T, F486S, and D1199N with altered spike positions. Later on, the booster dose or breakthrough infections contributed to elevating the immune profile. Several other mutations in BA.1.1-N460K, R346T, K444T, and BA.2.75.2-F486S have also conferred the neutralization resistance. The least studied T-cell response in SARS-CoV-2 affects HLA- TCR interactions, thus, it plays a role in limiting the virus clearance. Antigenic cartographic analysis has also shown Omicron’s drift from its predecessor variants. The rapidly evolving SARS-CoV-2 variants and subvariants have driven the population-based immunity escape in fully immunized individuals within short period. This could be an indication that Omicron is heading towards endemicity and may evolve in future with subvariants could lead to outbreaks, which requires regular surveillance. Full article

This study aimed to assess the association between the cycle threshold (Ct) values of SARS-CoV-2 and the risk of death from COVID-19 in adult patients from the Amazonas region of Peru during the circulation of the Lambda, Gamma, and Delta variants. The study population included both hospitalized and outpatient patients, symptomatic and asymptomatic, between August 2020 and August 2021. The standardized Ct values of the ORF1ab gene were categorized into low and high Ct groups based on the median Ct value (28.4). Mortality data within 60 days were obtained from the Peruvian epidemiological surveillance system (Notiweb). The risk of death was estimated using Cox regression, adjusting for relevant predictors and potential confounding variables. Among symptomatic COVID-19 patients, no significant difference in the risk of death was observed between those with low and high Ct values (adjusted hazard ratio [aHR] = 1.61; 95% confidence interval [CI], 0.97–2.67; p = 0.067). However, hospitalized patients with low Ct values had a significantly higher risk of death compared to those with high Ct values (aHR = 1.82; 95% CI, 1.06–3.12; p = 0.030). This association persisted after adjusting for age, sex, occupational group, symptom duration, comorbidities, and epidemic dynamics. In conclusion, while Ct values in symptomatic COVID-19 patients (both hospitalized and outpatient) are not associated with a 60-day mortality risk, a low Ct value is linked to an increased risk of death in hospitalized patients. Full article

Porcine epidemic diarrhea virus (PEDV) presents a substantial challenge to the global swine industry. However, the origin, host range, and potential cross-species transmission of PEDV remain poorly understood. This study characterizes a novel PEDV strain, CHFJFQ, isolated from diarrheic piglets in Fuqing, Fujian, China. Through sequencing and phylogenetic analysis, we determined that CHFJFQ belongs to the GIIa subgroup and is a recombinant with CH/HNXX/2016 as the major parent and NW17 as the minor parent. Compared to CV777, CHFJFQ exhibits multiple base deletions and insertions across the 5′UTR, ORF1a/b, S, and ORF3 genes. Phylogenetic analysis indicates shared ancestry with bat coronaviruses, though a direct zoonotic origin remains uncertain. Interestingly, CHFJFQ demonstrated its ability to infect human and mouse cell lines in vitro and, more significantly, caused in vivo infection in both pigs and mice. The primary target organs were the intestines, lungs, and spleen, resulting in 100% mortality in suckling piglets. PEDV CHFJFQ was detected in mouse tissues, but no clinical signs were observed, indicating limited cross-species pathogenicity. Overall, these findings offer crucial insights into the epidemiology, genetics, infectivity, and pathogenicity of PEDV and provide valuable information for vaccine development. Full article

Feline infectious peritonitis virus (FIPV) remains as one of the leading causes of morbidity and mortality in young cats from shelters and catteries worldwide. Since little is known about the molecular characteristics of currently circulating FIPV strains in Long Island, New York, samples from two shelter cats submitted to the Pathology Diagnostic Services of the Long Island University College of Veterinary Medicine, with gross and microscopic lesions consistent with those of FIP were processed for virus isolation, molecular characterization and full-length genome decoding. The younger shelter cat, a 1-year-old male (A15) was found dead without previous signs of illness. Postmortem examination revealed gross and microscopic lesions characterized by vasculitis, necrosis, hemorrhage, and pyogranulomatous inflammation confined to the colon and associated lymph nodes. The second cat, a 7-year-old spayed female (A37) had an identical clinical history and similar but widespread lesions, including fibrinous peritoneal effusion, cecal, colonic, renal, and hepatic involvement. The gross and microscopic diagnosis of FIP in these cats was confirmed by immunohistochemistry (IHC) demonstration of feline coronavirus antigen using mouse anti-FIPV3-70 monoclonal antibody. Virus isolation from saved frozen kidney and colon tissue was performed through several subsequent blind passages in MDCK and Vero cell lines. Confirmation of the FIPV isolation was done through qRT-PCR, IFA, western blot using N protein antibodies, and NGS of the full-length genome sequencing. The full-length genome sequences of the virus isolate from the two cats were decoded using next-generation sequencing (NGS) and deposited in the GenBank as accession numbers PQ192636 and PQ202302. The genome size of these isolates was (29355 and 29321) nucleotides (nt) in length, respectively. While their genome organization was consistent with other FIPV genomes as follows (5’UTR-ORF1ab-S-3abc-M-E-7b-3’UTR-3’), marked differential mutations were observed in the ORF1a/b, S, 3Abc, and 7b protein genes of the two FIPV isolates. One notable deletion of 34 nucleotides was observed in the 7b genes of one of these isolates but was absent in the other. We confirmed the potential recombination events during the evolution of those two FIPV field isolates with the potential parent virus as FECoV-US isolated in 1970 and the potential minor parent as the Canine coronavirus. Our results provide a comprehensive molecular analysis of two novel FIPV isolates causing fatal disease in shelter cats from Long Island. Diagnostic surveillance with molecular characterization and sequencing analysis of circulating FIPV strains within animal shelters may help early detect unique emerging clinical and pathological manifestations of the disease and develop more targeted prophylactic and therapeutic approaches to control it. Full article

Clustered regularly interspaced short palindromic repeats (CRISPR) molecular diagnostic technology is one of the most reliable diagnostic tools for infectious diseases due to its short reaction time, high sensitivity, and excellent specificity. However, compared with fluorescent polymerase chain reaction (PCR) technology, CRISPR molecular diagnostic technology lacks high-throughput automated instrumentation and standardized detection reagents for high sensitivity, limiting its large-scale clinical application. In this study, a high-throughput automated device was developed by combining reagent lyophilization, extraction-free technology, and a one-pot consumable system. This innovative approach enabled the rapid sample-in-result-out detection of 48 samples in 25 min and demonstrated high sensitivity and specificity for the qualitative analysis of clinical samples. The obtained results show that the detection limit of the designed system for African swine fever virus (ASFV) is 0.5 copies/μL. As a proof concept, a single-tube dual-target nucleic acid detection method was developed, achieving a detection limit of 5 copies/μL for the ORF1ab and N genes of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) within 45 min. The method is highly specific, reliable, and stable, providing a feasible solution for the clinical application of CRISPR nucleic acid detection technology. Full article

In this study, we present a novel face mask engineered for the collection of exhaled breath condensate (EBC) and its application and performance in a clinical study of COVID-19 infection status assessment versus the gold standard polymerase chain reaction (PCR) nasopharyngeal swab testing. EBC was collected within a clinical trial of COVID-19-infected and non-infected patients and analyzed by reverse transcription quantitative (RT-q) PCR, with the results being compared with nasopharyngeal sampling of the same patient. The cycle threshold (Ct) values of the nasopharyngeal samples were generally lower than those of EBC, with viral loads in EBC ranging from 1.2 × 10⁴ to 5 × 10⁸ viral particles mL⁻¹ with 5 min of breathing. From the 60 clinical patients’ samples collected, 30 showed a confirmed SARS-CoV-2 infection. Of these 30 individuals, 22 (73%) had Ct values < 40 (representing the threshold for SARS-CoV-2 infectivity) using both amplification of ORF1a/b and the E-gene. The 30 EBC samples from non-infected participants were all identified as negative, indicating a 100% specificity. These first results encourage the use of the face mask as a noninvasive sampling method for patients with lung-related diseases, especially with a view to equipping the face mask with miniaturized sensing devices, representing a true point-of-care solution in the future. Full article

Since the first reported case of COVID-19 in December 2019, several SARS-CoV-2 variants have evolved, and some of them have shown higher transmissibility, becoming the prevalent strains. Genomic epidemiological investigations into strains from different time points, including the early stages of the pandemic, are very crucial for understanding the evolution and transmission patterns. Using whole-genome sequences, our study describes the early landscape of SARS-CoV-2 variants in central India retrospectively (including the first known occurrence of SARS-CoV-2 in Madhya Pradesh). We performed amplicon-based whole-genome sequencing of randomly selected SARS-CoV-2 isolates (n = 38) collected between 2020 and 2022 at state level VRDL, ICMR-NIRTH, Jabalpur, from 11899 RT-qPCR-positive samples. We observed the presence of five lineages, namely B.1, B.1.1, B.1.36.8, B.1.195, and B.6, in 19 genomes from the first wave cases and variants of concern (VOCs) lineages, i.e., B.1.617.2 (Delta) and BA.2.10 (Omicron) in the second wave cases. There was a shift in mutational pattern in the spike protein coding region of SRAS-CoV-2 strains from the second wave in contrast to the first wave. In the first wave of infections, we observed variations in the ORF1Ab region, and with the emergence of Delta lineages, the D614G mutation associated with an increase in infectivity became a prominent change. We have identified five immune escape variants in the S gene, P681R, P681H, L452R, Q57H, and N501Y, in the isolates collected during the second wave. Furthermore, these genomes were compared with 2160 complete genome sequences reported from central India that encompass 109 different SARS-CoV-2 lineages. Among them, VOC lineages Delta (28.93%) and Omicron (56.11%) were circulating predominantly in this region. This study provides useful insights into the genetic diversity of SARS-CoV-2 strains over the initial course of the COVID-19 pandemic in central India. Full article

Equine herpesvirus type 1 (EHV-1) causes rhinopneumonitis, abortion, and neurological outbreaks (equine herpesvirus myeloencephalopathy, EHM) in horses. EHV-1 also causes lethal encephalitis in small laboratory animals such as mice and hamsters experimentally. EHV-1 ORF76 is a homolog of HSV-1 US9, which is a herpesvirus kinase. Starting with an EHV-1 bacterial artificial chromosome clone of neuropathogenic strain Ab4p (pAb4p BAC), we constructed an ORF76 deletion mutant (Ab4p∆ORF76) by replacing ORF76 with the rpsLneo gene. Deletion of ORF76 had no influence on replication, cell-to-cell spread in cultured cells, or replication in primary neuronal cells. In Western blots of EHV-1-infected cell lysates, an EHV-1 US9-specific polyclonal antibody detected multiple bands ranging from 35 to 42 kDa. In a CBA/N1 mouse infection model following intranasal inoculation, the parent and Ab4p∆ORF76 revertant caused the same histopathology in the brain and olfactory bulbs. The parent, Ab4p∆ORF76, and revertant mutant replicated similarly in the olfactory mucosa, although Ab4p∆ORF76 was not transported to the olfactory bulbs and was unable to infect the CNS. These results indicated that ORF76 (US9) plays an essential role in the anterograde spread of EHV-1. Full article

Bats, with their virus tolerance, social behaviors, and mobility, are reservoirs for emerging viruses, including coronaviruses (CoVs) known for genetic flexibility. Studying the cophylogenetic link between bats and CoVs provides vital insights into transmission dynamics and host adaptation. Prior research has yielded valuable insights into phenomena such as host switching, cospeciation, and other dynamics concerning the interaction between CoVs and bats. Nonetheless, a distinct gap exists in the current literature concerning a comparative cophylogenetic analysis focused on elucidating the contributions of sequence fragments to the co-evolution between hosts and viruses. In this study, we analyzed the cophylogenetic patterns of 69 host–virus connections. Among the 69 host–virus links examined, 47 showed significant cophylogeny based on ParaFit and PACo analyses, affirming strong associations. Focusing on two proteins, ORF1ab and spike, we conducted a comparative analysis of host and CoV phylogenies. For ORF1ab, the specific window ranged in multiple sequence alignment (positions 520–680, 770–870, 2930–3070, and 4910–5080) exhibited the lowest Robinson–Foulds (RF) distance (i.e., 84.62%), emphasizing its higher contribution in the cophylogenetic association. Similarly, within the spike region, distinct window ranges (positions 0–140, 60–180, 100–410, 360–550, and 630–730) displayed the lowest RF distance at 88.46%. Our analysis identified six recombination regions within ORF1ab (positions 360–1390, 550–1610, 680–1680, 700–1710, 2060–3090, and 2130–3250), and four within the spike protein (positions 10–510, 50–560, 170–710, and 230–730). The convergence of minimal RF distance regions with combination regions robustly affirms the pivotal role of recombination in viral adaptation to host selection pressures. Furthermore, horizontal gene transfer reveals prominent instances of partial gene transfer events, occurring not only among variants within the same host species but also crossing host species boundaries. This suggests a more intricate pattern of genetic exchange. By employing a multifaceted approach, our comprehensive strategy offers a nuanced understanding of the intricate interactions that govern the co-evolutionary dynamics between bat hosts and CoVs. This deeper insight enhances our comprehension of viral evolution and adaptation mechanisms, shedding light on the broader dynamics that propel viral diversity. Full article

Novel SARS-CoV-2 variants have multiple mutations that may impact molecular diagnostics. The markedly conserved S2 subunit may be utilized to detect new variants. A comparison of 694 specimens (2019–2022) in Thailand using a commercial RT-PCR kit and the kit in combination with S2 primers and a probe was performed. Delayed amplification in ORF1ab was detected in one BA.4 omicron, whereas no amplification problem was encountered in the S2 target. There were no statistically significant differences in mean Ct value between the target genes (E, N, ORF1ab, and S2) and no significant differences in mean Ct value between the reagents. Furthermore, 230,821 nucleotide sequences submitted by 20 representative counties in each region (Jan–Oct 2022) have been checked for mutations in S2 primers and probe using PrimerChecker; there is a very low chance of encountering performance problems. The S2 primers and probe are still bound to the top five currently circulating variants in all countries and Thailand without mismatch recognition (Jun–Nov 2023). This study shows the possible benefits of detecting S2 in combination with simultaneously detecting three genes in a kit without affecting the Ct value of each target. The S2 subunit may be a promising target for the detection of SARS-CoV-2 variants with multiple mutations. Full article

During the SARS-CoV-2 pandemic, the Dr. Risch medical group employed the multiplex TaqPath^TM COVID-19 CE-IVD RT-PCR Kit for large-scale routine diagnostic testing in Switzerland and the principality of Liechtenstein. The TaqPath Kit is a widely used multiplex assay targeting three genes (i.e., ORF1AB, N, S). With emergence of the B.1.1.7 (Alpha) variant, a diagnostic flaw became apparent as the amplification of the S-gene target was absent in these samples due to a deletion (ΔH69/V70) in the Alpha variant genome. This S-gene target failure (SGTF) was the earliest indication of a new variant emerging and was also observed in subsequent variants such as Omicron BA.1 and BA4/BA.5. The Delta variant and Omicron BA.2 did not present with SGTF. From September 2020 to November 2022, we investigated the applicability of the SGTF as a surrogate marker for emerging variants such as B.1.1.7, B.1.617.2 (Delta), and Omicron BA.1, BA.2, and BA.4/BA.5 in samples with cycle threshold (Ct) values < 30. Next to true SGTF-positive and SGTF-negative samples, there were also samples presenting with delayed-type S-gene amplification (higher Ct value for S-gene than ORF1ab gene). Among these, a difference of 3.8 Ct values between the S- and ORF1ab genes was found to best distinguish between “true” SGTF and the cycle threshold variability of the assay. Samples above the cutoff were subsequently termed partial SGTF (pSGTF). Variant confirmation was performed by whole-genome sequencing (Oxford Nanopore Technology, Oxford, UK) or mutation-specific PCR (TIB MOLBIOL). In total, 17,724 (7.4%) samples among 240,896 positives were variant-confirmed, resulting in an overall sensitivity and specificity of 93.2% [92.7%, 93.7%] and 99.3% [99.2%, 99.5%], respectively. Sensitivity was increased to 98.2% [97.9% to 98.4%] and specificity lowered to 98.9% [98.6% to 99.1%] when samples with pSGTF were included. Furthermore, weekly logistic growth rates (α) and sigmoid’s midpoint (t₀) were calculated based on SGTF data and did not significantly differ from calculations based on comprehensive data from GISAID. The SGTF therefore allowed for a valid real-time estimate for the introduction of all dominant variants in Switzerland and Liechtenstein. Full article

A complete genome sequence of an avian coronavirus (AvCoV; 27,663 bp excluding 3′ poly(A) tail) was determined using nontargeted next-generation sequencing (NGS) of an oropharyngeal swab from a backyard chicken in a live bird market in Arusha, Tanzania. The open reading frames (ORFs) of the Tanzanian strain TZ/CA127/19 are organized as typical of gammaCoVs (Coronaviridae family): 5′UTR-[ORFs 1a/1b encoding replicase complex (Rep1ab) non-structural peptides nsp2-16]-[spike (S) protein]-[ORFs 3a/3b]-[small envelop (E) protein]-[membrane (M) protein]-[ORFs 4a/4c]-[ORFs 5a/5b]-[nucleocapsid (N) protein]-[ORF6b]-3′UTR. The structural (S, E, M and N) and Rep1ab proteins of TZ/CA127/19 contain features typically conserved in AvCoVs, including the cleavage sites and functional motifs in Rep1ab and S. Its genome backbone (non-spike region) is closest to Asian GI-7 and GI-19 infectious bronchitis viruses (IBVs) with 87.2–89.7% nucleotide (nt) identities, but it has a S gene closest (98.9% nt identity) to the recombinant strain ck/CN/ahysx-1/16. Its 3a, 3b E and 4c sequences are closest to the duck CoV strain DK/GD/27/14 at 99.43%, 100%, 99.65% and 99.38% nt identities, respectively. Whereas its S gene phylogenetically cluster with North American TCoVs and French guineafowl COVs, all other viral genes group monophyletically with Eurasian GI-7/GI-19 IBVs and Chinese recombinant AvCoVs. Detection of a 4445 nt-long recombinant fragment with breakpoints at positions 19,961 and 24,405 (C- and N-terminus of nsp16 and E, respectively) strongly suggested that TZ/CA127/19 acquired its genome backbone from an LX4-type (GI-19) field strain via recombination with an unknown AvCoV. This is the first report of AvCoV in Tanzania and leaves unanswered the questions of its emergence and the biological significance. Full article

Since, during the Coronavirus disease 19 (COVID-19) pandemic, a large part of the human population has become infected, a rapid and simple diagnostic method has been necessary to detect its causative agent, the Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2), and control its spread. Thus, in the present study, we developed a colorimetric reverse transcription-loop-mediated isothermal amplification (RT-LAMP) kit that allows the detection of SARS-CoV-2 from nasopharyngeal swab samples without the need for RNA extraction. The kit utilizes three sets of LAMP primers targeting two regions of ORF1ab and one region in the E gene. The results are based on the colorimetric change of hydroxynaphthol blue, which allows visual interpretation without needing an expensive instrument. The kit demonstrated sensitivity to detect between 50 and 100 copies of the viral genome per reaction. The kit was authorized by the National Administration of Drugs, Food and Technology (ANMAT) of Argentina after validation using samples previously analyzed by the gold standard RT-qPCR. The results showed a sensitivity of 90.6% and specificity of 100%, consistent with conventional RT-qPCR. In silico analysis confirmed the recognition of SARS-CoV-2 variants of concern (B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.427, and B.1.429), and lineages of the Omicron variant (B.1.1.529) with 100% homology. This rapid, simple, and sensitive RT-LAMP method paves the way for a large screening strategy to be carried out at locations lacking sophisticated instrumental and trained staff, as it particularly happens in regional hospitals and medical centers from rural areas. Full article

We developed a convenient method for amplifying the complete SARS-CoV-2 sequence using in-house RT-PCR without virus culture. Forty-one stored throat swabs and blood specimens were collected from eight SARS-CoV-2 infections at multiple time points. Total RNA was extracted using the QIAamp viral RNA mini kit and pooled for higher RNA levels. Only those positive specimens by commercial real-time RT-PCR (RT-qPCR) were selected and amplified by in-house RT-PCR for complete sequences, followed by sequencing. Phylogenetic trees and exploratory analyses were performed using MEGA 11 and Simplot 3.5.1 software. Swab samples had significantly higher total RNA concentrations than plasma (p < 0.01). Positive results were found mainly in swabs, but one was found in plasma. Successful gene amplification depended on Ct values (Ct < 38). A non-synonymous substitution was found in ORF1ab/Nsp3 (at NC045512.2 position 6312, C to A) and most spike protein mutations occurred in the S1 subunit (residues 14–685). The proposed method is time-saving and reliable for rapid genomic analysis. Increasing sample volume and pooling them for RNA extraction increases RNA concentration without culture. Combining nucleotide sequences from specific variable regions of the genome is more efficient than conventional methods. Full article