Viremia

Relevant literature was collected by searching the PubMed, Ovid, and the Armed Forces Pest Management Board Literature Retrieval System databases using combinations of search terms including Aedes aegypti, Stegomyia fasciata (previous name for Ae. aegypti), Aedes albopictus, dengue, experiment, import, incubation, transmission, temperature, and travel. We did not restrict the search based on time of publication or language. Further material was found by reviewing references from identified papers.
The moment when a mosquito becomes infectious is not directly observable, so observations of the EIP are restricted to the window between exposure(s) and transmission experiment(s), defined by a minimum and maximum EIP. For example, if a mosquito is shown to be infectious 10 days after exposure, the EIP must be between 0 and 10 days. If the same mosquito is tested at day 5 and does not transmit DENV at that time, the EIP is between 5 and 10 days. For each observation, the maximum EIP was defined as the time from the first infectious blood meal to the first successful transmission of DENV. If transmissibility was tested and never successful, the maximum EIP is unknown. The minimum EIP was the time from the last infectious blood meal to the last negative transmission experiment or zero if there were no negative transmission experiments.
Acceptable transmission assays involved the confirmation of transmission to a naïve individual as evidenced by the onset of dengue or by laboratory evidence of infection such as hemagglutination inhibition or plaque reduction neutralization assays. Because dengue is used as an indicator, there may be some false-negative tests resulting from asymptomatic infections. We initially assume that all negative tests are truly negative and revisit this assumption later.
Observations of the EIP were limited to those in which Ae. aegypti or Ae. albopictus were fed on viremic humans or non-human primates. We also excluded observations in which infection of the mosquito was attained by injection or by feeding on animal blood or artificial media seeded with DENV, as these may not realistically mimic natural transmission.
Temperature data were recorded for each EIP observation when available. For observations with no temperature data, we obtained temperature data for the location of the study at the time of year when the study was undertaken from the Climate Research Unit 30-year mean climatology dataset (CL 2.0) [27] . The available temperature data was used to calculate a spatially and seasonally matched mean temperature for each observation.
The IIP analysis was restricted to events in which humans became sick after being experimentally infected by Ae. aegypti or Ae. albopictus or after being naturally exposed to DENV within a defined period of time by travelling into or out of an area with ongoing DENV transmission. In this case, the end event, the onset of symptoms, was always observed, but the exact exposure time is only known in the case of experimental infections. In those cases, the IIP was directly observed and therefore uncensored. In other cases, the maximum and minimum IIP were defined as the time from the first and last potential exposures, respectively, to the onset of illness. For example, a traveler who became sick 3 days after returning from a 10-day trip may have been exposed at any time during the trip, so the IIP must be between 3 and 13 days.
Further ancillary data collected for the analyses included the serotype of virus when known. The data is available in Text S1.

An index of reservoir competence (C_i) was derived as the product of three factors: susceptibility (s), the proportion of birds that become infected as a result of exposure; mean daily infectiousness (i), the proportion of vectors that become infected per day; and duration (d) of infectiousness, the number of days that a bird maintains an infectious viremia (12 (link)). This simple equation can be expressed as C_i = s _* i _* d. Thus, the competence index indicates the relative number of infectious vectors that derive from a particular bird species and is calculated as a function of the viremia that develops after mosquito-borne infection. To produce these data, we used a threshold level of infectious viremia of 10^5.0 PFU/mL serum and estimated infectiousness of each bird’s viremia levels from a standard curve for infection of C. pipiens as a function of viremic titer derived from Turell et al. (13 (link)) (Appendix D).

SARS-CoV-2 infection was defined as a positive RT-PCR or antigenic test result for a nasopharyngeal sample for symptomatic patients, or as a positive serological test result for patients with no symptoms. The SARS-CoV-2 Ct value varied between patients. Viremia was not analyzed. The SARS-CoV-2 variants also differed between patients. In five patients, the viral variant was confirmed by molecular methods (two Alpha and three Delta variants). In the remaining patients, infection was suspected to be caused by the predominant variant in the country at the time of diagnosis (Our World in Data, 2023 ). 12 patients were infected between April 2020 and February 2021 when the variants of clades 20A and 20B, which replaced the original virus infecting humans (clade 19A), predominated. Four patients were infected in April–May 2021, when the variant of concern (VOC) Alpha (clade 20I, genetically confirmed in two) predominated. Three patients infected from November 2021 to January 2022 had genetically confirmed infections with the VOC Delta (clade 21A). Finally, three patients were infected in January 2022, when Omicron (clade 21M) was the predominant VOC in their countries of residence (Fig. 2 A and Table S1). For 15 patients, infection was confirmed to have resulted from household transmission from an infected relative (Table S1). In another two patients (P4 and P5), the infection was contracted from a visiting relative. The mode of infection of the remaining five patients is unknown. None of the patients were vaccinated against SARS-CoV-2 at the time of infection.

In vivo, pegIFNα treatment was started when HBV/HDV-1a-, HBV/HDV-1p-, or HBV/HDV-3-infected mice reached stable HBV and HDV viraemia levels. Mice received pegIFNα (Pegasys; Roche, Basel, Switzerland) twice a week subcutaneously (25 ng/g body weight)¹⁷ (link)^,21 (link) for 4 or 9 weeks. Mice were sacrificed 24 h after the last pegIFNα injection. Cultured primary human hepatocytes (PHHs) (isolated from an HBV/HDV-infected mouse) received IFNα (1,000 IU/ml; Roferon-A; Roche, Basel, Switzerland) starting 1 day after plating. The culture medium was changed twice a week.
More methods (e.g. generation and infection of mice, virological measurements, RNA in situ hybridisation) can be found in the supplementary information.

Demographic, clinical, and laboratory data were collected at baseline by review of the electronic medical record. Cirrhosis etiology was classified as hepatitis C (viremic vs. post-SVR), hepatitis B, alcohol-associated liver disease, NAFLD, or other.20 Body mass index (BMI) was categorized according to World Health Organization classification: normal (BMI <25), overweight (BMI: 25–29.99), class I obesity (BMI: 30–34.99), class II obesity (BMI: 35–39.99), and class III obesity (BMI ≥40). Liver disease severity was assessed by the Child-Pugh class, with ascites and HE classified as none, mild or controlled, and severe or uncontrolled. Laboratory indices of interest included platelet count, aspartate transaminase, alanine transaminase, bilirubin, albumin, and INR. For multivariable models, age was dichotomized at the median value (60 y), whereas laboratory values were dichotomized based on the upper limit of normal.
Ultrasound exams at each site were performed according to LI-RADS technical recommendations.21 Ultrasound exams were interpreted by abdominal radiologists per routine clinical care, and findings were abstracted from radiology reports. We recorded the number, maximum diameter, and location of any liver observations on each imaging study. For those who developed PLC, we recorded the method of detection (surveillance, incidental, and diagnostic) and tumor stage.
Patients were followed per institutional standard of care from the time of index imaging until progression to PLC, death, liver transplantation, or end of follow-up (date of last available CT or MRI imaging), whichever occurred earliest. We documented the receipt and imaging findings of follow-up imaging (ultrasound, CT, or MRI) or other diagnostic evaluation (eg, receipt of liver biopsy) after the index liver observation. For patients who underwent liver transplantation, we recorded explant findings, including the presence of PLC, dysplastic nodules, or any other potential pathologic correlates of interest.