Dengue: A review of laboratory diagnostics in the vaccine age.

Background. Dengue is an important arboviral infection of considerable public health significance. It occurs in a wide global belt within a variety of tropical regions. The timely laboratory diagnosis of Dengue infection is critical to inform both clinical management and an appropriate public health response. Vaccination against Dengue virus is being introduced in some areas.Discussion. Appropriate diagnostic strategies will vary between laboratories depending on the available resources and skills. Diagnostic methods available include viral culture, the serological detection of Dengue-specific antibodies in using enzyme immunoassays (EIAs), microsphere immunoassays, haemagglutination inhibition or in lateral flow point of care tests. The results of antibody tests may be influenced by prior vaccination and exposure to other flaviviruses. The detection of non-structural protein 1 in serum (NS1) has improved the early diagnosis of Dengue and is available in point-of-care assays in addition to EIAs. Direct detection of viral RNA from blood by PCR is more sensitive than NS1 antigen detection but requires molecular skills and resources. An increasing variety of isothermal nucleic acid detection methods are in development. Timing of specimen collection and choice of test is critical to optimize diagnostic accuracy. Metagenomics and the direct detection by sequencing of viral RNA from blood offers the ability to rapidly type isolates for epidemiologic purposes.Conclusion. The impact of vaccination on immune response must be recognized as it will impact test interpretation and diagnostic algorithms.

Large-volume RT-LAMP enables extraction-free amplification of HIV RNA from fingerstick plasma.

BACKGROUND: Point-of-care (POC) nucleic acid amplification tests (NAAT) can significantly expand testing coverage, which is critical for infectious disease diagnostics and monitoring. The development of various isothermal amplification techniques greatly simplifies NAATs, but the cumbersome nucleic acid extraction step remains a bottleneck for the POC. Alternatively, extraction-free amplification, where crude samples are directly added into the assay, substantially simplifies the workflow. However, sample dilution is often needed in extraction-free amplification to reduce assay inhibition from sample matrices. Since NAATs are typically run at small volumes around 20 µL, the input sample quantity is therefore limited, resulting in an inevitable sensitivity loss. RESULTS: Here we explore the potential to perform isothermal amplification in larger reaction volumes to accommodate larger sample quantities, thereby improving sensitivity in extraction-free amplification. We demonstrated the approach by developing large-volume reverse transcription loop-mediated isothermal amplification (RT-LAMP) for HIV RNA detection from fingerstick plasma. We found that LAMP at reaction volumes up to 1 mL maintained the same performance. We then identified plasma dilution conditions needed to maintain the limit of detection in RT-LAMP. Subsequently, using inactivated HIV virus, we showed the successful detection of 24 HIV RNA copies in a 500 µL RT-LAMP reaction in the presence of 20 µL plasma (fingerstick volumes), translating to a viral load of 1200 copies per mL. To reduce the increased reagent cost with expanded reaction volumes, we further identified lower-cost reagents with maintained assay performance. Moreover, we showed that large-volume LAMP, compared to 20 µL reactions, could tolerate higher concentrations of various inhibitors in the sample, such as albumin and GuSCN. SIGNIFICANCE AND NOVELTY: NAATs are conventionally conducted at small reaction volumes. Here we demonstrated that LAMP can be run at large reaction volumes (over 100 µL) with maintained assay performance, allowing sample inhibition to be mitigated while accommodating larger sample quantities. The same strategy of expanding reaction volumes could be applied to other isothermal amplification methods and various POC applications, to streamline test workflows and/or improve assay sensitivity.

Predictors of Nucleos(t)ide Analogues Discontinuation Relapse: Hepatitis B Virus RNA versus Hepatitis B Surface Antigen.

OBJECTIVE: To compare the predictive value of hepatitis B virus (HBV) RNA and HBsAg quantification upon discontinuation of nucleos(t)ide analogues (NAs) therapy for clinical and virological relapse in chronic hepatitis B (CHB). STUDY DESIGN: Observational study. Place and Duration of the Study: Department of Infectious Diseases and Hepatology, The Second Hospital of Shandong University, Jinan, China, from July 2014 to December 2020. METHODOLOGY: CHB patients received single NAs and discontinued treatment following appropriate standards. HBsAg quantification was conducted using the i2000 Chemiluminescent Immunoassay (CLIA) Analyser, while serum HBV RNA quantification was performed using specific RNA target capture and simultaneous amplification and testing. The main observational endpoints included virological relapse and clinical relapse. RESULTS: Eighty-one patients were recruited, with 15 patients achieving HBsAg loss at cessation. Twenty-nine individuals encountered virological relapse, while 13 patients experienced clinical relapse. Thirty-one patients achieved HBsAg <100 IU/ml at NAs cessation, among whom 26 achieved undetectable HBV RNA, while four patients suffered virological relapse (15.4%). Serum HBV RNA emerged as an independent determinant of virological relapse (HR 1.850), clinical relapse (HR 2.020), and HBsAg loss after NAs cessation (HR 0.138). The presence of HBsAg <100 IU/ml at cessation did not serve as a predictor for virological relapse and clinical relapse. CONCLUSION: Lower HBV RNA levels predict a better off-treatment response. Discontinuation of prolonged NAs therapy appears as a viable and safe choice for patients with undetectable HBV RNA. In comparison to HBV RNA, HBsAg <100 IU/ml at cessation did not show sufficient predictive value for virological relapse and clinical relapse. KEY WORDS: HBV RNA, Hepatitis B surface antigen, Chronic hepatitis B, Relapse.

Evaluating the mosquito vector range for two orthobunyaviruses: Oya virus and Ebinur Lake virus.

BACKGROUND: Mosquito-borne viruses cause various infectious diseases in humans and animals. Oya virus (OYAV) and Ebinur Lake virus (EBIV), belonging to the genus Orthobunyavirus within the family Peribunyaviridae, are recognized as neglected viruses with the potential to pose threats to animal or public health. The evaluation of vector competence is essential for predicting the arbovirus transmission risk. METHODS: To investigate the range of mosquito vectors for OYAV (strain SZC50) and EBIV (strain Cu20-XJ), the susceptibility of four mosquito species (Culex pipiens pallens, Cx. quinquefasciatus, Aedes albopictus, and Ae. aegypti) was measured through artificial oral infection. Then, mosquito species with a high infection rate (IR) were chosen to further evaluate the dissemination rate (DR), transmission rate (TR), and transmission efficiency. The viral RNA in each mosquito sample was determined by RT-qPCR. RESULTS: The results revealed that for OYAV, Cx. pipiens pallens had the highest IR (up to 40.0%) among the four species, but the DR and TR were 4.8% and 0.0%, respectively. For EBIV, Cx. pipiens pallens and Cx. quinquefasciatus had higher IR compared to Ae. albopictus (1.7%). However, the EBIV RNA and infectious virus were detected in Cx. pipiens pallens, with a TR of up to 15.4% and a transmission efficiency of 3.3%. CONCLUSIONS: The findings indicate that Cx. pipiens pallens was susceptible to OYAV but had an extremely low risk of transmitting the virus. Culex pipiens pallens and Cx. quinquefasciatus were susceptible to EBIV, and Cx. pipiens pallens had a higher transmission risk to EBIV than Cx. quinquefasciatus.

SARS-CoV-2 detection in pediatric dental clinic wastewater reflects the number of local COVID-19 cases in children under 10 years old.

This was the first longitudinal study to analyze dental clinic wastewater to estimate asymptomatic SARS-CoV-2 infection trends in children. We monitored wastewater over a 14-month period, spanning three major COVID-19 waves driven by the Alpha, Delta, and Omicron variants. Each Saturday, wastewater was sampled at the Pediatric Dental Clinic of the only dental hospital in Japan's Saitama Prefecture. The relationship between the weekly number of cases in Saitama Prefecture among residents aged < 10 years (exposure) and wastewater SARS-CoV-2 RNA detection (outcome) was examined. The number of cases was significantly associated with wastewater SARS-CoV-2 RNA positivity (risk ratio, 5.36; 95% confidence interval, 1.72-16.67; Fisher's exact test, p = 0.0005). A sample from Week 8 of 2022 harbored the Omicron variant. Compared to sporadic individual testing, this approach allows continuous population-level surveillance, which is less affected by healthcare seeking and test availability. Since wastewater from pediatric dental clinics originates from the oral cavities of asymptomatic children, such testing can provide important information regarding asymptomatic COVID-19 in children, complementing clinical pediatric data.

Vertical transmission of SARS-CoV-2 delta-variant in a preterm infant.

BACKGROUND: As SARS-CoV-2 continues to be relevant and cause illnesses, the effect of emerging virus variants on perinatal health remains to be elucidated. It was demonstrated that vertical transmission of SARS-CoV-2 is a relatively rare event in the original SARS-CoV-2 strain. However, very few reports describe vertical transmission related to the delta-variant. CASE PRESENTATION: We report a case of a preterm male neonate born to a mother with positive SARS-CoV-2 and mild respiratory complications. The neonate was born by cesarean section due to fetal distress. The rupture of the amniotic membrane was at delivery. The neonate had expected prematurity-related complications. His nasopharyngeal swabs for RT-PCR were positive from birth till three weeks of age. RT-ddPCR of the Placenta showed a high load of the SARS-CoV-2 virus with subgenomic viral RNA. RNAscope technique demonstrated both the positive strand of the S gene and the orf1ab negative strand. Detection of subgenomic RNA and the orf1ab negative strand indicats active viral replication in the placenta. CONCLUSIONS: Our report demonstrates active viral replication of the SARS-CoV-2 delta-variant in the placenta associated with vertical transmission in a preterm infant.

The myriad roles of RNA structure in the flavivirus life cycle.

As positive-sense RNA viruses, the genomes of flaviviruses serve as the template for all stages of the viral life cycle, including translation, replication, and infectious particle production. Yet, they encode just 10 proteins, suggesting that the structure and dynamics of the viral RNA itself helps shepherd the viral genome through these stages. Herein, we highlight advances in our understanding of flavivirus RNA structural elements through the lens of their impact on the viral life cycle. We highlight how RNA structures impact translation, the switch from translation to replication, negative- and positive-strand RNA synthesis, and virion assembly. Consequently, we describe three major themes regarding the roles of RNA structure in flavivirus infections: 1) providing a layer of specificity; 2) increasing the functional capacity; and 3) providing a mechanism to support genome compaction. While the interactions described herein are specific to flaviviruses, these themes appear to extend more broadly across RNA viruses.

A Highly Sensitive Molecular Technique for RNA Virus Detection.

Zika (ZIKV) and Chikungunya (CHIKV) viruses are mosquito-transmitted infections, or vector-borne pathogens, that emerged a few years ago. Reliable diagnostic tools for ZIKV and CHIKV-inexpensive, multiplexed, rapid, highly sensitive, and specific point-of-care (POC) systems-are vital for appropriate risk management and therapy. We recently studied a detection system with great success in Mexico (Villahermosa, state of Tabasco), working with human sera from patients infected with those viruses. The research conducted in Mexico validated the efficacy of a novel two-step rapid isothermal amplification technique (RAMP). This approach, which encompasses recombinase polymerase amplification (RPA) followed by loop-mediated isothermal amplification (LAMP), had been previously established in the lab using lab-derived Zika (ZIKV) and Chikungunya (CHIKV) viruses. Crucially, our findings confirmed that this technique is also effective when applied to human sera samples collected from locally infected individuals in Mexico.

Condensation of RNase L promotes its rapid activation in response to viral infection in mammalian cells.

Oligoadenylate synthetase 3 (OAS3) and ribonuclease L (RNase L) are components of a pathway that combats viral infection in mammals. Upon detection of viral double-stranded RNA (dsRNA), OAS3 synthesizes 2'-5'-oligo(A), which activates the RNase domain of RNase L by promoting the homodimerization and oligomerization of RNase L monomers. Activated RNase L rapidly degrades all cellular mRNAs, shutting off several cellular processes. We sought to understand the molecular mechanisms underlying the rapid activation of RNase L in response to viral infection. Through superresolution microscopy and live-cell imaging, we showed that OAS3 and RNase L concentrated into higher-order cytoplasmic complexes known as dsRNA-induced foci (dRIF) in response to dsRNA or infection with dengue virus, Zika virus, or West Nile virus. The concentration of OAS3 and RNase L at dRIF corresponded with the activation of RNase L-mediated RNA decay. We showed that dimerized/oligomerized RNase L concentrated in a liquid-like shell surrounding a core OAS3-dRIF structure and dynamically exchanged with the cytosol. These data establish that the condensation of dsRNA, OAS3, and RNase L into dRIF is a molecular switch that promotes the rapid activation of RNase L upon detection of dsRNA in mammalian cells.

Predictive role of early treatment dynamics of HBV RNA and HBcrAg for HBeAg seroconversion in children with chronic hepatitis B.

This study aimed to assess the predictive capacity of emerging serological markers, serum HBV RNA and HBcrAg, for HBeAg seroconversion in children with HBeAg-positive chronic hepatitis B (CHB). Treatment-naïve HBeAg-positive CHB children who admitted to the Liver Disease Center of Hunan Children's Hospital between April 2021 and September 2022 and received treatment with the combined entecavir and interferon-alpha treatment were recruited. Serum HBV RNA and HBcrAg were measured at baseline and Weeks 12, 24, and 48 of treatment. Our study showed that serum HBV RNA (HR = 0.71, 95% CI: 0.56-0.91, p = 0.006), HBcrAg (HR = 0.60, 95% CI: 0.43-0.84, p = 0.003), and HBsAg (HR = 0.49, 95%CI: 0.36-0.69, p < 0.001) at Week 12 were independent predictors of HBeAg seroconversion. ROC curve analysis presented that serum HBV RNA decline value (ΔHBV RNA) at Week 36 and HBcrAg decline value (ΔHBcrAg) at Week 12 (AUC = 0.871, p = 0.003 and AUC = 0.810, p = 0.003, respectively) could effectively predict HBeAg seroconversion. Furthermore, the optimal critical values were determined and the children with ΔHBV RNA > 3.759 log10 copies/mL at Week 36 or ΔHBcrAg >0.350 log10 U/mL at Week 12 more likely to achieve HBeAg seroconversion. The serum HBV RNA and HBcrAg provide new insights into the treatment of CHB in children. Early assessment of serum HBV RNA and HBcrAg during treatment can assist clinical decision-making and optimize individualized therapeutic approaches.

Direct transmission of severe fever with thrombocytopenia syndrome virus from farm-raised fur animals to workers in Weihai, China.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease. SFTS virus (SFTSV) is transmitted by tick bites and contact with the blood or body fluids of SFTS patients. Animal-to-human transmission of SFTS has been reported in Japan, but not in China. In this study, the possible transmission route of two patients who fed and cared for farm-raised fur animals in a mink farm was explored. METHOD: An epidemiological investigation and a genetic analysis of patients, animals and working environment were carried out. RESULTS: It was found that two patients had not been bitten by ticks and had no contact with patients infected with SFTS virus, but both of them had skinned the dying animals. 54.55% (12/22) of the farm workers were positive for SFTS virus antibody. By analyzing the large, medium and small segments sequences, the viral sequences from the two patients, animals and environments showed 99.9% homology. CONCLUSION: It is suspected that the two patients may be directly infected by farm-raised animals, and that the virus may have been transmitted by aerosols when skinning dying animals. Transmission by direct blood contacts or animal bites cannot be ignored.

Clinical study of 400mg efavirenz treatment in newly diagnosed patients with HIV/AIDS.

The efficacy of 400mg efavirenz (EFV) once daily is reported to be similar to that of 600mg EFV. However, EFV-related toxic and side effects of 400mg EFV are significantly reduced. Here, the feasibility of reducing EFV to 400mg once a day in HIV-infected/AIDS patients was evaluated. Fifty patients were included. Patients were given 3TC+TDF+400mg EFV (n=25) or 3TC+TDF+600mg EFV (n=25). The proportion of patients with HIV RNA < 40 copies/mL and the adverse events served as the primary and secondary outcomes, respectively. HIV inhibition rates of the 3TC+TDF+400mg EFV group and 3TC+TDF+600mg EFV group were both 56.52% at week 24 and respectively 100%, 91.3% at week 48. During 48 weeks, 27 cases of adverse events were reported in the 3TC+TDF+400mg EFV group, lower than those in the 3TC+TDF+600mg EFV group, which had 39 cases. Compared with the 3TC+TDF+400mg EFV group, the incidence of transaminase, dizziness, hyperlipidemia and rashes all increased in the 3TC+TDF+600mg EFV group (P>0.05). No serious adverse events of the central nervous system occurred. The incidence of depression, sleep disturbance, and vertigo were similar (P>0.05). The efficacy of 400mg EFV is comparable to 600mg EFV. However, patients receiving 400mg EFV have fewer adverse events.

Isolation and characterization of a novel rodent hepevirus in long-tailed dwarf hamsters (Cricetulus longicaudatus) in China.

Hepeviruses have been identified in a broad range of animal hosts, including mammals, birds, and fish. In this study, rodents (n=91) from seven different species and ten pikas (Ochotona curzoniae) were collected in Qinghai Province, China. Using transcriptomic sequencing and confirmatory molecular testing, hepeviruses were detected in 27 of 45 (60 %) long-tailed dwarf hamsters (Cricetulus longicaudatus) and were undetected in other rodents and pika. The complete genome sequences from 14 representative strains were subsequently obtained, and phylogenetic analyses suggested that they represent a novel species within the genus Rocahepevirus, which we tentatively designated as Cl-2018QH. The virus was successfully isolated in human hepatoma (Huh-7) and murine fibroblast (17 Cl-1) cell lines, though both exhibited limited replication as assayed by detection of negative-sense RNA intermediates. A129 immunodeficient mice were inoculated with Cl-2018QH and the virus was consistently detected in multiple organs, despite relatively low viral loads. In summary, this study has described a novel rodent hepevirus, which enhances our knowledge of the genetic diversity of rodent hepeviruses and highlights its potential for cross-species transmission.

Evaluations of modes of pooling specimens for COVID-19 screened by quantitative PCR and droplet digital PCR.

Though pooling samples for SARS-CoV-2 detection has effectively met the need for rapid diagnostic and screening tests, many factors can influence the sensitivity of a pooled test. In this study, we conducted a simulation experiment to evaluate modes of pooling specimens and aimed at formulating an optimal pooling strategy. We focussed on the type of swab, their solvent adsorption ability, pool size, pooling volume, and different factors affecting the quality of preserving RNA by different virus solutions. Both quantitative PCR and digital PCR were used to evaluate the sampling performance. In addition, we determined the detection limit by sampling which is simulated from the virus of different titers and evaluated the effect of sample-storage conditions by determining the viral load after storage. We found that flocked swabs were better than fibre swabs. The RNA-preserving ability of the non-inactivating virus solution was slightly better than that of the inactivating virus solution. The optimal pooling strategy was a pool size of 10 samples in a total volume of 9 mL. Storing the collected samples at 4 °C or 25 °C for up to 48 h had little effect on the detection sensitivity. Further, we observed that our optimal pooling strategy performed equally well as the single-tube test did. In clinical applications, we recommend adopting this pooling strategy for low-risk populations to improve screening efficiency and shape future strategies for detecting and managing other respiratory pathogens, thus contributing to preparedness for future public health challenges.

Genomic Analysis of Monkeypox Virus During the 2023 Epidemic in Korea.

We aimed to characterize the genomes of monkeypox virus isolates from the Far East, providing insights into viral transmission and evolution. Genomic analysis was conducted on 8 isolates obtained from patients with monkeypox virus disease in the Republic of Korea between May 2022 and early 2023. These isolates were classified into Clade IIb. Distinct lineages, including B.1.1, A.2.1, and B.1.3, were observed in 2022 and 2023 isolates, with only the B.1.3 lineage detected in six isolates of 2023. These genetic features were specific to Far East isolates (the Republic of Korea, Japan, and Taiwan), distinguishing them from the diverse lineages found in the Americas, Europe, Africa, and Oceania. In early 2023, the prevalence of the B.1.3 lineage of monkeypox virus identified in six patients with no overseas travel history is considered as an indicator of the potential initiation of local transmission in the Republic of Korea.

Accelerated diagnosis: a crosslinking catalytic hairpin assembly system for rapid and sensitive SARS-CoV-2 RNA detection.

The COVID-19 pandemic has underscored the urgent need for rapid and reliable strategies for early detection of SARS-CoV-2. In this study, we propose a DNA nanosphere-based crosslinking catalytic hairpin assembly (CCHA) system for the rapid and sensitive SARS-CoV-2 RNA detection. The CCHA system employs two DNA nanospheres functionalized with catalytic hairpin assembly (CHA) hairpins. The presence of target SARS-CoV-2 RNA initiated the crosslinking of DNA nanospheres via CHA process, leading to the amplification of fluorescence signals. As a result, the speed of SARS-CoV-2 diagnosis was enhanced by significantly increasing the local concentration of the reagents in a crosslinked DNA product, leading to a detection limit of 363 fM within 5 min. The robustness of this system has been validated in complex environments, such as fetal bovine serum and saliva. Hence, the proposed CCHA system offers an efficient and simple approach for rapid detection of SARS-CoV-2 RNA, holding substantial promise for enhancing COVID-19 diagnosis.

Molecular phylogeny and secondary structure analysis of hop stunt viroid (HSVd) associated with Mulberry (Morus alba) in India.

Hop stunt viroid (HSVd), a small, single stranded, circular, non-coding infectious RNA known to cause infection in various economically important crop plants. In the present investigation, a study was conducted in the southern part of Karnataka districts of India to detect the possible association of HSVd infection in mulberry plants. A total of 41 mulberry plants showing typical viroid-like symptoms along with asymptomatic samples were collected and screened using conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using a specific set of HSVd-Fw/ HSVd-Re primers. Out of 41 samples, the study confirmed the presence of HSVd in six samples of mulberry collected from Ramanagara (1 sample), Chikkaballapur (3 samples) and Doddaballapura (2 samples) regions with an expected HSVd amplicon size of ∼ 290-300 nucleotides. The mechanical transmission of HSVd was also confirmed on cucumber (cv. Suyo) seedlings through bioassay, which was reconfirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative nucleotide sequences were deposited in the NCBI GenBank. Subsequently, molecular phylogenetic analysis showed that HSVd mulberry isolates from this study were most closely related to grapevine isolates, indicating a common origin. On the other hand, it was shown to belong to a different group from mulberry isolates so far reported from Iran, Italy, Lebanon, and China. The secondary structure analysis of HSVd mulberry Indian isolates exhibited substitutions in the terminal left, pathogenicity, and variable regions compared to those of the Indian grapevine isolates. As far as this study is concerned, HSVd was detected exclusively in some mulberry plants with viral-like symptoms, but the pathogenesis and symptom expression needs to be further investigated to establish the relationship between HSVd and the disease symptoms in the mulberry plants.

Method for the Enrichment of N6-Methyladenosine-Modified Cellular and HIV-1 RNA.

N6-methyladenosine (m6A) modification of RNA is an important area in studying viral replication, cellular responses, and host immunity. HIV-1 RNA contains multiple m6A modifications that regulate viral replication and gene expression. HIV-1 infection of CD4+ T-cells or HIV-1 envelope protein treatment upregulates m6A levels of cellular RNA. Changes in the m6A modification of cellular transcripts in response to HIV-1 infection provide new insights into the mechanisms of posttranscriptional gene regulation in the host cell. To better investigate the functions of m6A modification in HIV-1 infection and innate immune responses, it is helpful to standardize basic protocols. Here, we describe a method for the selective enrichment of m6A-modified RNA from HIV-1-infected primary CD4+ T-cells based on immunoprecipitation. The enriched RNA with m6A modifications can be used in a variety of downstream applications to determine the methylation status of viral or cellular RNA at resolution from transcript level down to single nucleotide.

Structures of H5N1 influenza polymerase with ANP32B reveal mechanisms of genome replication and host adaptation.

Avian influenza A viruses (IAVs) pose a public health threat, as they are capable of triggering pandemics by crossing species barriers. Replication of avian IAVs in mammalian cells is hindered by species-specific variation in acidic nuclear phosphoprotein 32 (ANP32) proteins, which are essential for viral RNA genome replication. Adaptive mutations enable the IAV RNA polymerase (FluPolA) to surmount this barrier. Here, we present cryo-electron microscopy structures of monomeric and dimeric avian H5N1 FluPolA with human ANP32B. ANP32B interacts with the PA subunit of FluPolA in the monomeric form, at the site used for its docking onto the C-terminal domain of host RNA polymerase II during viral transcription. ANP32B acts as a chaperone, guiding FluPolA towards a ribonucleoprotein-associated FluPolA to form an asymmetric dimer-the replication platform for the viral genome. These findings offer insights into the molecular mechanisms governing IAV genome replication, while enhancing our understanding of the molecular processes underpinning mammalian adaptations in avian-origin FluPolA.