Human papillomavirus (HPV) is responsible for almost all of the 570,000 new cases of cervical cancer and approximately 311,000 deaths per year. HPV vaccination is an integral component of the World Health Organization’s (WHO) global strategy to fight the disease. However, high vaccine prices enforced through patent protection are limiting vaccine expansion, particularly in low- and middle-income countries. By limiting market power, patent buyouts could reduce vaccine prices and raise HPV vaccination rates while keeping innovation incentives. We estimate the global patent buyout price as the present discounted value (PDV) of the future profit stream over the remaining patent length for Merck’s HPV vaccines (Gardasil-4 and 9), which hold 87% of the global HPV vaccine market, in the range of US$ 15.6–27.7 billion (in 2018 US$). The estimated PDV of the profit stream since market introduction amounts to US$ 17.8–42.8 billion and the estimated R&D cost to US$ 1.05–1.21 billion. Thus, we arrive at a ratio of R&D costs to the patent value of the order of 2.5–6.8%. We relate this figure to typical estimates of the probability of success (POS) for clinical trials of vaccines to discuss if patent protection provides Merck with extraordinarily strong price setting power. Cervical cancer, a disease mainly caused by human papillomavirus (HPV), is the fourth most common type of cancer in women with approximately 570,000 new cases and 311,000 deaths per year globally , 5.The vast majority 95%) of deaths caused by cervical cancer and around half of the world’s new cases are in low-income countries (LIC) and middle-income countries (MIC) –9. The h5% of deaHigh vaccine prices, enabled by patent protection, is considered one of the main factors limiting the expansion of HPV vaccination in developing countries , 13. PhaThere are two strategies to promote vaccine R&D: pull programmes that provide financial reward to companies that develop successful vaccines and push programmes that provide direct funds for research. Pull programmes include prizes, compulsory licensing and patent buyouts whereas push programmes include research grants and tax credit . For lowet al. [et al. [This study focusses on patent buyouts by government agencies to supply medicines at lower costs and make them affordable to poorer countries via licensing production to many competitors . The litet al. . Our mea [et al.  and mark [et al. .www.clinicaltrial.gov and a literature search. We calculate the R&D costs based on previously estimated costs per subject [in vitro and in vivo studies) and clinical trials (phase I-III). Most of the pre-clinical development of Gardasil-4 was performed by the National Cancer Institute (NCI), Georgetown University and University of Queensland who were the first to develop virus-like particle (VLP) technology used in the vaccine in the early 1990s [We also estimate the global value of the Gardasil patents at market entry, using a similar methodology as for deriving the patent buyout price, and derive estimates of R&D costs by identifying each clinical trial sponsored by Merck on  subject , clinica subject . Vaccinely 1990s . Merck lly 1990s .We compute the ratio of estimated R&D costs for clinical trials to the patent value (PDV of the stream of profits from market entry to patent expiry). In theory, this ratio is the probability of success (POS) in finding an innovation that is required to just cover the R&D costs in expected value . We thusOur estimated global patent buyout in year 2020 for Gardasil vaccines is in the range of US$ 15.6–27.7 billion (in 2018 US$), depending on assumed real annual return to investment in alternative uses, price estimates, and cost estimates. The patent value, we estimate between US$ 17.8–42.8 billion. These high values reflect our findings of high price-cost margins which could be around 100 for the U.S. and China and still well above 10 in MIC. The estimated R&D costs for both Gardasil vaccines combined lie in the range between US$ 1.05–1.21 billion. These figures imply a ratio of R&D costs and the patent value between 2.5–6.8%. We will argue that this figure is considerably lower than we would expect from the estimated POS for vaccines from Phase I to approval in the literature, suggesting that the patent holder earns extraordinarily high profits.et al. [Estimates of R&D costs from confidential sources such as self-reported R&D costs from pharmaceutical companies and estimates of industry experts are impossible to assess for accuracy, representativeness, or sensitivity to outliers . Our estet al.  estimateHPV is a group that encompasses over 100 viruses and 15 of them have been shown to be responsible for almost all cases of cervical cancer: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 . One of The two most widely used prophylactic vaccines in the market are Gardasil-4  and Cervarix . Gardasil-4 was approved for both U.S. and European markets in September 2006 whereas Cervarix was approved in September 2007 , 31. TheVaccination is an integral part of the WHO global strategy to combat HPV infections and related diseases . AccordiIt is estimated that 70–80% of females in pre-pubertal age have to be vaccinated in order to achieve herd immunity . AlthougAcademic institutes in the U.S. and Australia first developed the technology employed in the VLP-based vaccines in the early 1990s. Merck and GSK then improved on the original invention and performed the subsequent steps required to bring the vaccine to the market . Merck’sThere is no centralized database to obtain information about the status of the various patents related to a particular vaccine in different countries/regions. In the U.S., Gardasil patents expire in 2028, and similarly in other advanced countries , 53. We The biggest factor that will drive the increase in global demand of HPV vaccine is the introduction of HPV vaccination in China, India and Indonesia (three of the four most populous countries in the world). These countries are expected to represent approximately 1/3 of the global market by 2030 , 54. Chia priori not evident that patents are stimulating innovation [Our focus on HPV vaccination derives, apart from data availability issues, from its importance for global health and the prevalence of HPV related diseases particularly in developing countries. Patents like those for HPV vaccines provide intellectual property protection that bars entry of generic competitors for an extended period of time, therefore awarding monopoly price setting power. Because inventions use prior knowledge that is protected by patents, it is novation . One of novation . WorkingUnder the agreement on trade-related aspects of intellectual property rights (TRIPS), administered by the World Trade Organization (WTO) and enforced in 1995, member countries with an industry capable of manufacturing vaccines must enforce patent protection of medicines and biological products . There iBrazil, India and China have a large generic pharmaceutical industry supplying 64% of vaccines purchased by UNICEF and 43% of vaccines procured by GAVI . In addiWHO has launched a global effort to eliminate cervical cancer by promoting introduction of HPV vaccination in all countries . AccordiAnother factor limiting the expansion of HPV vaccination are the high prices of HPV vaccines that are prohibitive to GAVI and PAHO supported LIC and MIC . To dateThe discussion strongly suggests that the entrance of new manufacturers is crucial to ensure sufficient supply and lower HPV vaccine prices, thus increasing static economic efficiency by mitigating price setting power of the incumbent . Given tDespite these arguments in favor of the patent buyout at the suggested price, we do not aim to calculate the net social benefit from the patent buyout because of several difficulties to predict the entry response of generic manufacturers and the price responses by the patent holder. First, for some period of time after the buyout, Merck would likely continue to be the major supplier and prices may remain significantly above marginal costs. It is also plausible that some LIC and MIC countries would choose to postpone introducing HPV vaccination  until after generics enter the market, if they knew it was imminent. Likewise, GAVI and the PAHO revolving fund would aim at negotiating lower prices.We first propose our methodology to estimate the patent buyout price and to assess the extent of price setting power in the market of HPV vaccination. We then discuss the data and assumptions for our estimations.T, i.e. year 2020 this gives us the patent buyout price (with caveats discussed in Section 4) for the vaccine Gardasil under the assumption of risk-neutrality. Arguably, large pharmaceutical companies like Merck can be considered risk-neutral as they are owned by shareholders that can hold diversified asset portfolios. Moreover, they have diversified product portfolios and can diversify R&D effort. By contrast, a  risk-averse company facing market uncertainty would agree to a patent buyout price that is lower than the PDV of expected future profits.Our first goal is to estimate the PDV of the expected future stream of real profits for the vaccine Gardasil, starting from year To calculate the time path of profits, we need estimates for sales prices, variable costs, and fixed costs. We assume that the variable cost per unit (dose) is independent of the number of produced doses such that total variable costs are proportional to the units sold. They consist of:user costs of physical capital ,costs for materials needed to express and purify the VLPs of the targeted HPV types,costs for filling and packaging ,costs for operating labor .Fixed costs consist of:overhead labor costs for managing and supervising the manufacturing process in the facilities used for producing the vaccine,labor costs for maintaining the facilities and equipment,marketing expenditure .et al. [et al. [We follow Clendinen et al.  to treat [et al.  by modelex ante and thus cannot be considered being part of the fixed cost [Legal costs for patent defence, patent infringement disputes, product liability litigations, commercial litigations, government proceedings or environmental matters should generally be considered as fixed costs. Merck gives a detailed account of these costs in its annual reports. However, so far, no legal proceedings or costs were reported for Gardasil-4 and Gardasil-9. Also notably, R&D expenditures are incurred xed cost .That said, we still miss some labor-intensive fixed cost components that are private information like building relations with suppliers of material, training costs and costs for developing the manufacturing to scale process and packaging design. There is reason to assume, however, that in the present context these costs are small. First, large scale manufacturing of VLP based vaccines was already being done in the 1990s and early 2000s for a variety of VLPs vaccines against HEV, influenza, BTV, rotavirus and PPV . All theWe also miss distribution costs. However, the vast majority of distribution costs are covered by government and NGO’s. In the U.S., the Centers for Disease Control (CDC) Vaccine Supply and Assurance Branch (VSAB) oversees all aspects of its vaccine purchase and distribution in the country . In the j∈{1,2,…,J} and denote the time horizon by T (U.S. patent expiry year 2028 in our application). We use information about the time paths of the predicted future number of doses sold at the regional level, real terms. In year t, operating profits from sales to country group j are then given bypjt and yjt denote sales prices and number of doses in region j and year t, respectively, and c the variable costs per dose.We index the country (group) in which Gardasil is sold by T onwards in country group j until time T asr is the real annual return to investment in alternative uses . We present our estimates for the annual real interest rate r in the range of 3–7%.Based on Eq No. t by ft and the PDV of the fixed cost stream from period T until time T byFixed costs are typically not region-specific, as production takes place in few facilities and the product is shipped from there. We denote real global fixed costs in year T for the considered regions is given byThus, the global patent buyout price in year c and fixed costs F are the same for Gardasil-4 and Gardasil-9.We assume that both variable unit cost T at the PDV of future profits, V(T), fully compensates the patent holder for its profit losses associated with lower price setting power and is thus incentive-compatible.Arguably, buying out a patent in We also aim to estimate the patent value from the perspective of the date introduced in the market and compare it with R&D costs. Gardasil-4 was introduced in September 2006, i.e. 2007 was the first full year Gardasil was sold. To generate conservative estimates of the patent value and since we do not have price information for 2006, we neglect profits for 2006 and include marketing costs for 2006 in total fixed costs for 2007.s≤T, the total patent value is given by V(s). If the market for innovations is characterized by free entry and firms are risk-neutral, the expected value of an innovation  equals R&D costs [μ denote the POS in the innovation process, such that μ∙V(s) is the expected patent value . A firm is incentivized to conduct the innovation project as long as μ∙V(s)≥R&Dcosts. The POS required to break even in expected value is given when the inequality is binding. It is defined asDenoting the first full year of market introduction &D costs . Formallμ0 with typical estimates of the average POS for clinical trials of other vaccines reported in the literature. Higher price setting power would increase V(s) and thus decrease μ0. A value of μ0 that is considerably lower than the average POS found for other vaccines could therefore indicate excessive price setting power of the patent holder.Our data allows us to estimate the right-hand side of Eq No. μ0, we sum up both the PDV of profits from both Gardasil-4 and Gardasil-9 since market introduction until patent expiry when computing V(s), the denominator of Eq No. In our context of HPV vaccines, the POS is the cumulative probability of moving from the three phases of clinical trials to approval. Recall that Gardasil-9 was an improvement of Gardasil-4, the first VLP-based HPV vaccine approved by the FDA . GardasiWe now outline the data sources, how we extrapolate missing information, and how we deal with measurement problems and uncertainty with respect to future values. We convert all monetary variables to 2018 US$, using the U.S. consumer price index . Detailepjt), regional number of doses sold (yjt), and variable unit cost (c). The prices of HPV vaccines vary greatly depending on procurement agreement and countries’ income [According to Eq No. ’ income . Merck a’ income , 71. Whe’ income , 72. In ’ income . The med’ income , 74. The’ income , US$ 4.5’ income  and in-b’ income  and Indo’ income  , GAVI  estimateWe account for measurement problems of past vaccine doses’ sales and uncertainty in the predictions for future sales by creating a “low” and “high” scenario by deducting and adding 20% of the sales of doses sold to the middle estimates that we derive by the outlined procedure  adjuvant. The manufacturing process has four steps: cultivation, extraction, purification type-specific VLPs from L1-recombinant producer cells, filling and packaging . Associaet al.    expenditure to promote Gardasil-4 and Gardasil-9 in the U.S. since market entry from ‘The Nielsen Company’. We use their additional information that DTCA spending in the U.S. is 90% of the global spending and create two scenarios for the missing marketing expenditure of health care providers (S11 Table in V(s), according to Eqs No. www.clinicaltrial.gov to identify the Gardasil related clinical trials sponsored by Merck in each phase. To calculate the costs on subjects  for each clinical phase we use the phase-specific estimates of costs per subject in Light et al. [www.clinicaltrial.gov. In addition to subject-related costs, there are site costs (for recruitment and retention of subjects as well as administrative and site monitoring) and study costs  . We add 10–15% to the subject-related, site and study costs to account for R&D capital costs.According to Based on the assumptions and data presented in Section 2.3, It is interesting to look at price-cost margins. Even based on the high estimate for variable costs, the mark-up factor (price divided by unit variable costs) in 2016 is around 100 for the U.S. and China, twelve in MIC and almost three for sales mediated by GAVI.r = 0.03, r = 0.05 and r = 0.07. We estimate the PDV of future operating profits from the perspective of year 2020 in the range of US$ 16.45–28.94 billion, and the total PDV of the stream operating profits from the perspective of year 2007 in the range of US$ 19.82–44.25 billion. Reflecting the comparatively high sales prices in the U.S., half of the future profits are earned in the U.S. The profit share earned in the U.S. is even higher when considering the time period 2007–2028, as the sales share in the U.S. market was higher in the past than predicted for the future, reflecting market expansion to less developed countries.We derive two estimates for regional operating profits. The “high” estimate combines the 20% upward deviation from the middle estimates of the number of doses in et al. [We next derive the PDV of fixed costs by applying Eq No. et al.  and on tRecently, Merck announced an expansion of the North Carolina facility and build a new one, the two facilities are expected to be fully operational from 2022 . Since tBased on DTCA spending data for the Gardasil vaccines in the U.S. , the assumptions on marketing spending on health providers, and the annual fixed manufacturing costs in Tables Assuming that global marketing spending on health providers is 50% and 150% of global DTCA spending for the “low” and “high” estimate, respectively, implies that the PDV of the stream of global marketing costs from the perspective of 2007 is in the range of US$ 1.11–2.12 billion (in 2018 US$). It exceeds the PDV of manufacturing fixed costs for the period 2007–2028 considerably (US$ 140–330 million) and is at least as high as R&D costs, as will become apparent in Section 3.4. We estimate that the PDV of future manufacturing costs from the perspective of 2020 amounts to US$ 149–270 million. It exceeds the PDV of the stream of marketing costs in the period 2020–2028 , reflecting that marketing spending has sharply declined some years after market introduction. The PDV of total future fixed costs from the perspective of 2020 is in the range of US$ 213–393 million. For the entire patent length, the PDV of the total fixed cost stream from the perspective of 2007 amounts to US$ 1.25–2.45 billion.Using Eq No. We next relate the total value of the Gardasil patents in In phase I of clinical trials the vaccine is tested in a small number of healthy individual to identify the best route to administer the vaccine, frequency and dose escalation, the maximum tolerated dose (MTD), and side effects . The maiet al. [et al. [Light et al.  estimate [et al. , the sitAs shown in μ0 in Eq No. μ0 = 0.025. For the upper bound, dividing the high estimate for R&D costs  and the low estimate of the patent value  implies μ0 = 0.068.We finally put our estimates for total R&D costs in relation to the estimates of the total value of the Gardasil patents. This provides us with a range for the innovation probability that is required to cover R&D costs in expected value of the patents, as defined by μ0? In a sample of more than 1,800 trials at phase I in the 2000s, Wong et al. [μ0, suggesting that the Gardasil patents generate extraordinarily high profits.What do we know about the POS for vaccines targeting infectious diseases to which we can compare our estimate for g et al.  found a g et al. . This sug et al. . In sum,Patents are an effective strategy to prevent new entrants in the market. The high number of patent applications for HPV vaccines by Merck and GSK in emerging countries with a large pharmaceutical industry such as China, Brazil and India shows that it is a strategy commonly implemented . Patent We estimated that the remaining patent buyout value for Gardasil supplied by Merck in 2020 as the PDV of the future stream of expected profits in the range of US$ 15.6–27.7 billion (in 2018 US$), depending on the assumed interest rate for discounting and the estimated ranges for the number of demanded doses, variable costs, and fixed costs. On the one hand, the estimated remaining private patent value may be viewed as an upper bound for the patent buyout price since the original manufacturer Merck would still be able to make profits after the patent buyout because of its brand name and its established production capacity. On the other hand, given the private information of the patent holder, we may want to propose a sufficiently high patent buyout price that is unlikely to fall short of the remaining patent value, to incentivize the patent holder to agree to the patent buyout. In sum, albeit calculating the net social benefit of the patent buyout is beyond the scope of this paper, the suggested range for the patent buyout price may be viewed as a benchmark for negotiating an agreement with the patent holder.While the global patent buyout of Gardasil we suggest is incentive-compatible for the patent holder, it is a financially challenging proposal. A preferable albeit not necessarily successful strategy to reduce prices could be to reach a licensing agreement with Merck to allow generic manufacturing of Gardasil for LIC and MIC. The Medicines Patent Pool (MPP), a United Nations-backed global health organization, negotiates with pharmaceutical companies and other global health stakeholders (i.e. governments and NGOs) licensing to allow generic companies to manufacture low cost medicines for LIC and MIC . This paIn order to assess the price setting power of Merck in providing its HPV vaccines, we have also estimated both the R&D costs and the global patent value, which is the PDV of the profit stream for the period 2007–2028. The ratio of these figures is the POS needed to break even in expected value. Using information on clinical trials, we estimated the R&D costs for the Gardasil innovation to be around US$ 1.05–1.21 billion, while the global patent value amounts to US$ 17.8–42.8 billion. The implied R&D to patent value ratio of 2.5–6.8% is below the average POS in clinical trials for vaccines found in the literature. As a caveat and suggestion for future research, making the point that the current patent system generates excessive price setting power, i.e. is more generous than needed to elicit desirable R&D effort, would require estimations of the relationship between R&D cost and the patent value for many more pharmaceuticals. Availability of such information may alter price negotiations for prescription drugs in health systems and may ultimately call for adjustment of the patent system.S1 File(DOCX)Click here for additional data file.


Pigeon paramyxovirus type 1 (PPMV-1) infection causes high morbidity in pigeons, resulting in a significant burden to the poultry industry. In this study, we isolated three PPMV-1 strains from diseased pigeons collected in Guangdong Province, South China, from June 2017 to April 2019. Genetic analysis revealed that these three PPMV-1 strains and most of the PPMV-1 strains isolated from China after 2011 were clustered into sub-genotype VI.2.1.1.2.2. Our Bayesian analysis revealed that the VI.2.1.1.2.2 viruses might have originated in Europe. Phylogeographic analyses revealed that East and South China might have played a key role in seeding the VI.2.1.1.2.2 PPMV-1 epidemic in China. To characterize the effect of age at infection on the outcome of PPMV-1 infection in pigeons, we investigated the pathogenesis and transmission of the pigeon/Guangdong/GZ08/2017 (GZ08) virus in 3-, 6-, and 12-week-old pigeons. Two of six 12-week-old pigeons inoculated with GZ08 survived, and all of the 3- and 6-week-pigeons inoculated with GZ08 died. Moreover, the GZ08 virus could be transmitted to 3-, 6-, and 12-week-old naïve contact pigeons. The lethality of the GZ08 virus through contact with 3-, 6-, and 12-week-old pigeons was 100%, 66.7%, and 0%, respectively, suggesting that the transmissibility of the GZ08 virus was stronger in young pigeons. These findings demonstrated that East and South China was the epicenter for dissemination of VI.2.1.1.2.2 PPMV-1, and age at infection has an impact on the outcome of PPMV-1 infection in pigeons. Orthoavulavirus of the family Paramyxoviridae and the subfamily Avulavirinae. The NDV genome is a single-stranded, negative-sense RNA, coding six structural proteins, including nucleoprotein, phosphoprotein, matrix protein, fusion (F) protein, hemagglutinin–neuraminidase and large protein, and two non-structure proteins, V and W [Newcastle disease (ND) is a worldwide infectious disease that causes significant economic losses to the poultry industry. Its causative agent is the virulent Newcastle disease virus (NDV), a member of the genus  V and W . Accordi V and W , NDV can V and W .Genotype VI NDVs, also referred to as pigeon paramyxovirus type 1 (PPMV-1) viruses, are variant strains of NDV associated with infections of Columbiformes, including wild and domestic pigeons and doves. Since PPMV-1 was first identified in the Middle East, particularly in Iraq, in 1978 , they haAlthough most PPMV-1 strains are nonpathogenic to chickens, they can cause high morbidity and mortality in pigeons, leading to a degree of economic loss. The primary clinical signs in pigeons after being infected with PPMV-1 are neurological signs, such as paralysis, torticollis, and a twisted neck and head ,14. The In the current study, we isolated three PPMV-1 strains from diseased pigeons collected in Guangdong Province, from June 2017 to April 2019, and investigated the prevalence, molecular evolution, and phylogeography of these viruses. Moreover, we also characterized the effect of age at infection on the outcome of PPMV-1 infection in pigeons and analyzed the antigenic variation between epidemical PPMV-1 in South China and the commercial vaccine LaSota strain.Animals were treated humanely and with regard for alleviation of suffering. All animal experiments were approved by the South China Agricultural University Experimental Animal Welfare Ethics Committee .g for 10 min, the supernatants were propagated in 10-day-old specific-pathogen-free (SPF) chicken embryos. The presence of NDVs was confirmed via hemagglutination (HA), HA-inhibition (HI) assay, and reverse transcription polymerase chain reaction (RT-PCR), as described previously [The PPMV-1 strains were isolated from diseased pigeons suspected of having PPMV-1 infection in Guangdong Province, South China, from June 2017 to April 2019. Respiratory and neurological signs, including gasping and twisting of the head and neck, were observed in these diseased pigeons. The collected tissue samples were homogenized in 1.0 mL of phosphate-buffered saline (PBS) supplemented with antibiotics  using TissueLyser II . After centrifugation at 5000× eviously . After tThe virus genome was isolated from infective allantoic fluid using RNeasy Mini Kit (Qiagen) and transcribed into cDNA with M-MLV (Moloney Murine Leukemia Virus) Reverse Transcriptase . PCR amplification was performed using 12 pairs of overlapping primers specific to the complete genomes of NDV, as described previously . To deteA total of 364 complete F gene sequences of PPMV-1 submitted before 31 December 2019, were collected from GenBank. All sequences were aligned by the version 7.058 program. A maximum likelihood  tree wasIn order to determine the temporal structure of all the PPMV-1 strains in the VI.2.1.1.2.2, a regression of root-to-tip genetic distance was performed for these sequences using the TempEst software  based onTo understand the spatial diffusion patterns of VI.2.1.1.2.2 PPMV-1 strains, the ancestral geographical regions, diffusion rates, and migration patterns of these viruses were analyzed by the asymmetric continuous-time Markov chain phylogeographic model with the Bayesian Stochastic Search Variable Selection (BSSVS) in the BEAST version 1.10.4 software . Due to Three-week-old pigeons were purchased from a commercial pigeon farm  and housed in isolators under negative pressure with food and water provided ad libitum. None of the pigeons had any history of disease or vaccination against NDV. Prior to animal experimentation, all pigeons were confirmed to be seronegative for NDV antibodies by the HI (Hemagglutination Inhibition) test with GZ08 antigens and negative for NDV in oropharyngeal and cloacal swabs by RT-PCR, as described previously .6 egg infectious dose (EID50) of the GZ08 virus in a 200-μL volume. Eight hours after inoculation, three 3-week-old pigeons, inoculated with 200 μL PBS via the same route, were cohoused with these inoculated pigeons as the naïve contact group. The pigeons in the control group (n = 10) were inoculated intranasally with 200 μL PBS. At 3 and 7 days post-infection (DPI), we euthanized three inoculated pigeons and determined the virus titers in the brain and lungs. To detect virus shedding, oropharyngeal and cloacal swabs were collected at 3, 5, 7, 9, 11, 13, 17, and 21 DPI and suspended in 1000 μL PBS with antibiotics . All collected samples were stored at −80 °C until further use. Virus titers were determined by EID50 assay, as described previously [Twelve 3-week-old pigeons were inoculated intranasally with 10eviously . At 21 DThree-week-old pigeons were housed in isolators until they were 6 and 12 weeks old. The procedures conducted on 6- and 12-week-old pigeons followed the same design as that for 3-week-old pigeons.R-value was calculated using the formula from a previous report [The cross-reactivity HI assay between the commonly used vaccine LaSota and the PPMV-1 strain, GZ08, was conducted as described previously . Brieflys report .p < 0.05 and p < 0.01 were considered to indicate statistically significant differences, unless stated otherwise.Statistical analysis was performed using GraphPad Prism version 8.0.1 software . Differences of virus titers in 3-, 6-, and 12-week-old pigeons were compared by one-way analysis of variance (ANOVA). Compared to the 3-week-old pigeon group, After passage in SPF chicken eggs, three PPMV-1 strains were identified by HA/HI and RT-PCR assays, designated as pigeon/Guangdong/GZ08/2017 (GZ08), pigeon/Guangdong/SZ12/2018, and pigeon/Guangdong/HY25/2019 under GenBank numbers MN893303–MN893305.Based on the classification system for assigning NDVs proposed by , an ML tR2 were 0.8864 and 0.6175, respectively, confirming the presence of structure in the data. Next, the best model was estimated by comparing the marginal likelihood estimates, using PS and SS. The results demonstrated that the prior use of a strict clock and Bayesian skyline coalescent tree provided the best data fit . The rate estimated from the root-to-tip regression (1.46 × 10−3 subs/site/year) was also similar to the results of the Bayesian analysis.The root-to-tip regression analysis of the VI.2.1.1.2.2 viruses showed that the correlation coefficient and data fit . The meaThe time-scaled MCC tree of VI.2.1.1.2.2 PPMV-1 isolates based on the complete F gene showed that all VI.2.1.1.2.2 PPMV-1 viruses isolated from China were generally divided into groups I–II . Groups To illustrate the population size of sub-genotype VI.2.1.1.2.2 NDVs, the Bayesian skyline coalescent was reconstructed to reveal that the relative genetic diversity of sub-genotype VI.2.1.1.2.2 NDVs. As shown in To estimate the global circulation of the VI.2.1.1.2.2 PPMV-1 strains, eight geographic regions  were divided according to geographical location, and a BSSVS procedure was implemented . Europe,All of 3- and 6-week-old pigeons inoculated with GZ08 died within 12 and 13 DPI, respectively, whereas two of the six 12-week-old pigeons that were inoculated survived. In 3-week-old inoculated pigeons, mild clinical signs such as depression were observed at 4 DPI; one pigeon died, and four pigeons exhibited severe clinical signs, including twisting of the head and neck, incoordination, and paralysis, at 5 DPI . Clinica50, respectively, and these values increased to 5.83 and 5.42 log10 EID50, respectively, at 7 DPI. In 6-week-old pigeons, the mean viral loads in the brain and lungs were 1.58 and 2.5 log10 EID50, respectively, at 3 DPI. In 12-week-old pigeons, the mean viral loads in the brain and lungs were 1.83 and 3.91, respectively, at 3 DPI, and 4.5 and 4.67 log10 EID50, respectively, at 7 DPI. Notably, the virus titers in the brain were significantly higher than those in 6- or 12-week-old infected pigeons (p < 0.05). At 7 DPI, virus titers of the tested tissues of all the inoculated pigeons, except the 3-week-old group, increased, compared with those at 3 DPI. Generally, virus titers were higher in 3-week-old pigeons than in 6- and 12-week-old pigeons for the investigated time periods.To determine the replication of the GZ08 virus in 3-, 6-, and 12-week-old pigeons, three inoculated pigeons were euthanized at 3 and 7 DPI. The virus was detectable in the brain and lungs of 3-, 6-, and 12-week-old pigeons . In 3-weTo determine virus shedding in inoculated pigeons of different ages, oropharyngeal and cloacal swabs were collected from all chickens at 3, 5, 7, 9, 11, 13, 17, and 21 DPI. Shedding of the GZ08 virus could be detected in both oropharyngeal and cloacal swabs of inoculated 3-, 6-, and 12-week-old pigeons during the experimentation period .To determine the intraspecific horizontal transmission of PPMV-1, three pigeons served as the naïve contact group and were cohoused with inoculated pigeons in each group. The naïve contact 3-week-old pigeons began to show clinical signs at 8 DPI, and their mortality was 100% . HoweverR value was 0.597, indicating minor antigenic differences between the GZ08 strain and the commonly used vaccine strain LaSota.To analyze the antigenic variation of PPMV-1 and the commercial vaccine strain LaSota, a cross-reactive HI assay was performed. The Since PPMV-1 was first identified in China in 1985 , PPMV-1 According to the MCC tree of the VI.2.1.1.2.2 PPMV-1, the viruses isolated from Europe were located at the root of the tree with the highest root state posterior probability of 0.96, indicating that Europe might have been the origin of VI.2.1.1.2.2 viruses. For the precise of the analysis, we excluded single data in India or Egypt. The deduction was further supported by the migration link from Europe to East China in the diffusion processes of VI.2.1.1.2.2 PPMV-1. Previous studies demonstrated that the European continent was the epicenter of global dissemination of PPMV-1 due to the movement of pigeons ,40. Cai −3 subs/site/year , and similar rates were observed in Miller et al.’s [−3 (strict) and 1.70 × 10−3 (relaxed), and Ramey et al.’s [−3 (strict) and 1.32 × 10−3 (relaxed) for wild-bird origin class I NDVs, and 1.02 × 10−3 (strict) and 1.10 × 10−3 (relaxed) for class II NDVs. However, the substitution rate of VI.2.1.1.2.2 PPMV-1 viruses was shown to be faster than those of virulent genotype II (7.05 × 10−5) and IX (2.05 × 10−5) NDVs in a recent study [In our Bayesian analysis, the estimated substitution rate of the F gene from VI.2.1.1.2.2 PPMV-1 viruses was 1.317 × 10et al.’s  report fet al.’s  report, nt study . NotablyA previous study found that PPMV-1 virus led to a 100% mortality in 1-day-old chickens by intratracheal and intracerebral inoculation, whereas no death was observed in 14-day-old chickens after being infected via the same route . HoweverR value between the GZ08 strain and the commonly used vaccine LaSota was 0.59, indicating a minor antigenic difference between LaSota and GZ08. The results were similar to those of previous research [The cross-reactivity HI assay showed that the research ,34. A laresearch ,52,53,54research . TherefoIn conclusion, we isolated three VI.2.1.1.2.2 PPMV-1 from Guangdong Province, from June 2017 to April 2019, and demonstrated that East and South China was the epicenter for dissemination of VI.2.1.1.2.2 PPMV-1. We also showed that the age at infection has an impact on the outcome of PPMV-1 infection in pigeons. To control PPMV-1 infection in pigeons, it is necessary to develop antigenically matched vaccines and to strengthen the biosecurity measures that prevent viruses from infecting pigeons.


The preauricular sinus (PAuS) is a congenital foramen, opening or invagination, usually located on the crus of the auricular helix and is considered a congenital malformation and component of multiple syndromes. The structure can be present unilaterally or bilaterally, with the possibility of more than one fistula present on one ear, predominantly on the auricular tags. As a well-defined and established clinical entry, PAuS has a very strictly laid-out history. However, different works of art give us a glimpse into the structure before its first true clinical description, showing that the PAuS was known to man long before it was first clinically described, such as those of Hieronymous Bosch, with the first medical descriptions being attributed to Heusinger and Virchow. In modern times, the condition is considered both an individual malformation and a component of several genetic syndromes. The preauricular sinus (PAuS) is a congenital malformation characterized by a dent, dimple, or a foramen, usually located on the crus of the auricular helix -2. RarelThe structure can be present unilaterally or bilaterally, with the possibility of more than one fistula present on one ear, predominantly on the auricular tags -6. The sAs a well-defined and established clinical entry, PAuS has multiple contributors in the medical field that helped define it and its significance. However, different works of art give us a glimpse into the structure before its first true clinical description, showing that the PAuS was known to man long before it was first clinically described.Artistic representationHieronymus Bosh c. 1450-1516) was a Dutch artist whose most famous painting is “The Garden of Earthly Delights”. The painting illustrates exterior and interior panels . The ext450-1516 First medical descriptionsThe PAuS was first described in 1864 by C. F. Heusinger when describing the findings in a patient characteristic of the brachio-oto-renal (BOR) syndrome . He detaFollowing Heusinger's description, Rudolph Virchow 1821-1902) also described the structure in 1864, although simply stating in his article “I also know a patient like that” . Virchow1-1902 alSir James Paget also published on the subject in 1878, describing patients with such fistulae present not only on their ears but also on their neck . Based oDuring the first part of the 20th century, the PAuS became known as “natural earring holes” and “fistula auris congenita”, with several authors publishing on the subject, including its pattern of inheritance ,15,16. TOne of the most important contributions from that time period was in 1955 by Fourman and Fourman, who researched the inheritance of PAuS, constructing a genealogical tree, focusing on the inheritance of the structure . They coDespite that initial interest, however, little to no articles were published after the late 1940s, until a small resurgence in later years, where the structure reappears under a new name - PAuS. Since then, research on the structure has seldom been carried out, and the mechanisms of its transgenerational inheritance remain widely forgotten in the literature of the 1930s and 1940s.PAuS-associated syndromesMelnick-Fraser SyndromeThe syndrome was first described in 1864 by Heusinger . HoweverBeckwith-Wiedemann SyndromeThe condition was originally described in 1963 by American pathologist John Beckwith as a combination of exomphalos, macroglossia, and gigantism, therefore referred to as exomphalos-macroglossia-gigantism (EMG) syndrome . In 1964, however, independently of John Beckwith, the German pediatrician Hans-Rudolph Wiedemann also described several patients with the same hallmarks of the disease but also included symptoms such as adrenal hyperplasia . Over tiLachiewicz-Sibley SyndromeFirst described in 1985 by Lachiewicz et al., this is one of the rarest syndromes ever described . The synTwenty-first centuryToday although still relatively under-researched, the PAuS is a common clinical entity with well-defined clinical strategies for treatment. Some studies have identified a genetic association between PAuS and the 8q11.1-13.1 locus; however, the results have not been reproduced widely ,7,38. OtThe clinical course has been well described, and treatment strategies including predominantly surgical excision have been well established in modern otorhinolaryngology and head and neck surgery ,7,8,40. The PAuS, as it was first described, is often a component of several eponymous inherited syndromes such as Melnik-Fraser (BOR syndrome), Beckwith-Wiedemann, and Lachiewicz-Sibley, which also include a variety of constant kidney and other distant variable malformities such as cysts, fistulae, facial and neural defects, and others. These syndromes relatively rarely underline the importance of Virchow’s original statement that the condition is caused by malfusion of pharyngeal arches, a process taking place parallel to some stages of kidney development. However, there is no evidence in the scientific literature inferring that the presence of PAuS always concludes to the presence of any of the aforementioned syndromes. Although the exact model of inheritance of the PauS, according to modern understandings and its genetic and molecular hallmarks, are yet to be fully determined and described, some authors still consider it as an irregular dominant mutation with reduced penetrance.