Novel oral poliovirus vaccine type 2 (nOPV2) is being developed to reduce the rare occurrence of disease and outbreaks associated with the genetic instability of the Sabin vaccine strains. Children aged 1 to 5 years were enrolled in two related clinical studies to assess safety, immunogenicity, shedding rates and properties of the shed virus following vaccination with nOPV2 (two candidates) versus traditional Sabin OPV type 2 (mOPV2). The anticipated pattern of reversion and increased virulence was observed for shed Sabin-2 virus, as assessed using a mouse model of poliovirus neurovirulence. In contrast, there were significantly reduced odds of mouse paralysis for shed virus for both nOPV2 candidates when compared to shed Sabin-2 virus. Next-generation sequencing of shed viral genomes was consistent with and further supportive of the observed neurovirulence associated with shed Sabin-2 virus, as well as the reduced reversion to virulence of shed candidate viruses. While shed Sabin-2 showed anticipated A481G reversion in the primary attenuation site in domain V in the 5’ untranslated region to be associated with increased mouse paralysis, the stabilized domain V in the candidate viruses did not show polymorphisms consistent with reversion to neurovirulence. The available data from a key target age group for outbreak response confirm the superior genetic and phenotypic stability of shed nOPV2 strains compared to shed Sabin-2 and suggest that nOPV2 should be associated with less paralytic disease and potentially a lower risk of seeding new outbreaks. Despite this progress, the spread of circulating type 2 vaccine-derived polioviruses (cVDPV2) represents a major threat to the complete eradication of poliovirus. More specifically, reduced population immunity against the type 2 strain in OPV-using countries following cessation of routine use of type 2 OPV in 2016 precipitated an increase in cVDPV2 circulation, with evidence that targeted responses to cVDPV2 with Sabin OPV type 2 (mOPV2) seeded new outbreaks in adjacent areas5. The number of acute flaccid paralysis cases associated with cVDPV2 has increased from two in two countries in 2016, to 366 in 16 countries in 2019, and then to over 1000 in 24 countries in 2020 (https://polioeradication.org/polio-today/polio-now).Tremendous progress has been made towards the eradication of poliovirus, with both wild type 2 and type 3 polioviruses eradicated and type 1 endemic in only two countries8.The underlying genetic mechanism for the evolution of Sabin-2 into VDPV2 is well documented, with key early steps in the loss of attenuation involving reversions in domain IV (U398C) and domain V (A481G) of the internal ribosome entry site within the 5’ untranslated region (UTR) and in amino acid 143 of VP1 capsid protein11. Both candidates include a genetically stabilized domain V termed S15, which was designed to attenuate the strain to a similar degree as the Sabin strains, while avoiding the possibility of loss of attenuation via single nucleotide changes. More specifically, the potential for thermodynamic strengthening of the RNA structure via point mutations was reduced by eliminating the use of U-G base pairs12.Two novel OPV2 candidates have recently been developed and advanced through Phase 1 and 2 clinical studies with the goal of providing similar protection as mOPV2 but with reduced risk of loss of attenuation13. The novel OPV2 candidate 2 (nOPV2-c2) supplements the S15 domain V with codon deoptimization of the capsid region to further attenuate the strain14.In addition to the S15 domain V, novel OPV2 candidate 1 (nOPV2-c1) also includes relocation of the cis-acting replication element (cre) to the 5’ untranslated region (UTR) of the virus in order to protect the S15 domain V from replacement through a single recombination event. In addition, the nOPV2-c1 includes two modifications to the 3D polymerase which reduce the frequency of recombination events and improve replication fidelity15. In infants, nOPV2-c1 induced non-inferior seroprotection rates compared to Sabin OPV2 at both 105 and 106 CCID50 (50% cell culture infectious dose) doses, while nOPV2-c2 met the non-inferiority criterion at the 106 CCID50 dose but not at the 105 CCID50 dose11.Both candidates were shown to be generally safe and well tolerated in Phase 1 and 2 clinical studies9. Here, this paper reports the first use of this workflow to provide a head-to-head comparison of shed nOPV2 candidates with shed Sabin-2 from two clinical trials in Panamanian children.In addition to having an acceptable clinical safety profile and non-inferior immunogenicity, the novel vaccines are intended to retain their attenuated phenotype in a manner superior to Sabin OPV2. Details of a workflow to examine the genetic heterogeneity and neurovirulence of fecally-shed virus using next-generation sequencing and mouse neurovirulence assay have recently been described9.Samples for evaluation of genetic stability of shed virus following administration of monovalent OPV2 (mOPV2) or nOPV2 candidates were collected from 1-to-5-year-old children in Panama during phase 4 and phase 2 trials . A modified transgenic mouse neurovirulence test (mTgmNVT) was utilized to confirm whether the attenuation phenotype of nOPV2 candidates was maintained in shed virus. In addition, to assess the genetic stability of the genome in shed virus, next-generation sequencing (NGS) was conducted. NGS and mTgmNVT were used to evaluate virus shed in a subset of stool samples  from clinical trial participants, as described in METHODS50/g stool11, the threshold for EES selection. Notably, the vast majority of 1-to-5-year-old participants in the M5 trial were previously vaccinated with IPV and/or bOPV, and only 10% had received tOPV.The M2 and M5 studies were conducted non-contemporaneously, with the global cessation of OPV2 usage in routine immunization occurring between the two study periods. As such, due to differences in prior polio vaccination regimens, children in the included age group had varying levels of baseline intestinal immunity. As shown in Table As shown in Table Descriptive Fig.  and compp = 1.0000), with estimated mean paralysis rate (across both mouse genders) of 32.2% .For mOPV2, five EES  were tested Fig. . Two EESp = 0.9827), with estimated mean paralysis rate of 0.03% .For nOPV2-c1, 22 EES  had valid results, with two EES paralyzing a single mouse each and no paralysis for the remaining 20 EES (range 0 to 11.1%). Descriptive model-based analysis indicates the absence of a detectable mouse gender effect (p = 0.0023), with overall estimated mean paralysis rate of 8.8% .Similarly, for nOPV2-c2, 29 EES (one per participant ranging from days 2 to 21) had valid results. Paralysis rates ranged from 0 to 80%, with six EES paralyzing ≥ 40% of mice (discussed further under Next-generation sequencing of shed virus). Descriptive model-based analysis indicates lower paralysis rates for female versus male mice (p = 0.9789), and significantly reduced odds of mouse paralysis from virus obtained from nOPV2-c1 recipients compared to mOPV2 recipients  , and a significant (p = 0.0425) reduction in the estimated odds of mouse paralysis from virus obtained from nOPV2-c2 recipients compared to mOPV2 recipients .Results of the comparative model-based analysis indicate the absence of a detectable mouse gender effect (60) Fig. . There iNGS was performed on full-length poliovirus genome following PCR amplification of virus present in stool and cell culture-amplified virus from the selected stool samples. Descriptive analysis of the frequency of polymorphisms observed by NGS in key attenuating regions of Sabin-2  or attenuating and modified regions of the nOPV2 shed virus was conducted to assess retention of the engineered modifications, and determine if the sequence variations in shed virus correlate with the observed neurovirulence results in the transgenic mouse model.In addition, coverage maps were used to determine if any co-infection  was observed in the EES. Following NGS, the sample reads were mapped to a type 2 reference  as well as to Sabin 1 and 3 references. Non-polio enteroviruses, Sabin-1, and Sabin-3 were not detected in any of the EES evaluated by NGS and no recombinant viruses were observed.As expected, shed Sabin-2 virus reverted rapidly (by day 7). NGS data Fig.  support NGS was performed directly on virus in stool (two replicates of each EES stool) and on virus cell-culture amplified from stool, where available, for 31 nOPV2-c1 participants. Polymorphisms noted in stool were also largely observed in the culture-amplified virus (used in the mTgmNVT), and there was strong correlation between the single nucleotide polymorphisms (SNPs) observed directly in stool versus amplified virus, demonstrating minimal bias from the cell-culture amplification process. Supplementary Fig. 9. SNPs in the relocated cre at nucleotide 123 and/or 179, which strengthens U-G base pair to C-G or U-A were observed in all EES day 4 to 28 at variable levels up to 100% frequency. Several other SNPs were observed in the relocated cre region at low levels, although U172A was observed at higher levels in the stools of a day 9 EES. In addition, several samples accumulated C121U and/or A181G polymorphisms at <10% average frequency in samples when present. These polymorphisms allow an additional base pair at 121-181 through formation of U-A or C-G pair, likely extending the stem length of cre5.Mutations in the modified regions and known attenuating mutations are summarized in Fig. Reversion in domain V (nucleotides 468-535) of the 5’ UTR is the main determinant for restoration of virulence (as measured in the mouse neurovirulence model) following mOPV2 administration in humans. No polymorphisms consistent with increased virulence were detected in S15 domain V of shed nOPV2-c1. Although several SNPs were observed at low levels , none are pair-forming or pair-strengthening. Within the 5’ UTR, variation was also noted at position 459 in domain IV, corresponding to nucleotide 398 in Sabin-2, which is known to have minor impact on attenuation (modest increase in neurovirulence).Mutations that result in reversion of the unprotected secondary attenuation site, VP1-143 (e.g. T2970C coding for VP1-143T), were observed consistently in the EES, often at consensus or higher levels.No mutations impacting the modified Rec1 or Hifi3 locations of the 3D polymerase were observed, but a few low-level silent polymorphisms in the cre knockout region were observed . One mutation in the cre KO regions was associated with an amino acid change, although it was observed only in single replicates of the day 5, 6 and 9 EES.13.Two samples with limited paralysis (one mouse each) had high levels of VP1-I143T and some strengthening of the cre 123/179 pair; one also had U459C reversion . These polymorphisms were previously shown to have an additive impact on neurovirulence in the mouse model16. For example, the level of VP3-E234K in the lot used in this study was 45%. As anticipated from research studies on molecular clones containing this mutation, there was no consistent pressure for or against this mutation. VP1-E295K (G3425A) frequencies were nearly always less than or equal to VP1-N171D (A3053G), and they are often present at similar levels in the EES  and culture-amplified virus where available for 38 participants who received nOPV2-c2. Polymorphisms in the modified regions and known attenuating sites are summarized in Fig. As shown in Fig. Five polymorphisms were observed in domain V (described further below in S15 Domain V polymorphisms), although none are known to be associated with reversion to virulence. C486U at consensus level was observed in a day 6 EES. While not possible to assess for co-location, levels are very similar to U2909C mutation yielding VP1-I143T (73% versus 75%) suggesting the possibility that the domain V polymorphism is a carrier mutation selected due to co-location on a genome with VP1-143T.There were no mutations in the modified CpG sites in the P1 region meeting the reporting algorithm described in METHODS, although several changes were observed in the other regions of the genome (Supplementary Table 50) compared to nOPV2-c2 (Table 50 of 3.86).To further characterize the virulence of the shed nOPV2-c2 viruses, the 6 EES with paralysis rate ≥40% were evaluated in multi-dose neurovirulence tests to determine the 50% paralytic dose . This is in contrast to four of the six nucleotides in this loop that are 100% conserved.In the nOPV2-c1 EES Fig. , C550U wFor nOPV2-c2, 5 SNPs were observed in domain V Fig. . C482U iThe most consequential safety risks associated with use of mOPV2 are rare cases of vaccine-associated paralytic poliomyelitis (VAPP) in vaccine recipients or their close contacts, and reversion and circulation of the vaccine strain in the community with emergence of vaccine-derived polioviruses in settings of persistently low immunization rates. A primary objective of nOPV2 development is to substantially reduce the potential for reversion to virulence, if not eliminate the risks posed by mOPV2. To address the rapidity and extent to which nOPV2 strains and Sabin-2 reacquire virulence in vaccinated children, their genetic stability was assessed using next-generation sequencing, and phenotypic stability was assessed using a mouse neurovirulence test on shed vaccine virus samples.19 and molecular clones with relevant mutations (e.g. A481G). In contrast, no changes that would be expected to contribute to loss of attenuation were observed in S15 domain V of the shed nOPV2 strains. Generally, although SNPs were observed in shed nOPV2 strains, they were at low levels and none led to strengthening of a base pair. The specific observed changes generally appeared to be neutral or destabilizing  and theoretically further attenuate the shed nOPV2 viruses.As expected, Sabin-2 virus reverted rapidly, with shed virus showing known key genetic reversions soon after vaccination and corresponding high paralysis rates in the mouse model once reverted. Although the number of samples available was small, the results were consistent with historic data for excreted Sabin-2 viruses16 showed anticipated behavior upon replication in vivo. For example, VP1-E295K—which causes a temperature-sensitive defect limiting replication at 37 °C—was selected against unless compensated for by the presence of a VP1-N171D mutation. VP1-I143X and VP1-N171D mutations, which increase mouse neurovirulence individually but not additively, were only exceptionally selected together in the same virus.The most noteworthy genetic changes observed in nOPV2 strains were mutations leading to (a) amino acid substitutions at VP1-143, a U398C polymorphism  in domain IV and (b), polymorphisms that strengthen a base pair in the relocated cis-acting replication element (cre) or extend the relocated cre in nOPV2-c1. These are potentially relevant to neurovirulence and viral fitness, though largely anticipated from prior work studying the evolution of the Sabin-2 and nOPV2 strains. Some mutations previously identified in the nOPV2-c1 vaccine lots and characterized phenotypically14. For shed nOPV2-c2 EES with paralysis rates of ≥40%, the multi-dose mouse assay provided PD50 estimates consistent with previous data using molecular constructs9. These showed that U398C or VP1-143V/T in the nOPV2-c2 clone contribute to virulence in the mouse neurovirulence model  using Tg66 mice. Combining the two mutations in one clone resulted in a more virulent virus (PD50 2.4), indicating an additive effect. Shed virus from all 6 EES show higher virulence than nOPV2-c2 (PD50 6.3) and Sabin-2 (PD50 5.9), although all are still considerably less virulent than Sabin-2 with reversion at 481 in domain V (PD50 1.8).The results from the mouse neurovirulence tests indicated a statistically significant reduction of the paralysis rate for shed virus from nOPV2-c1 or nOPV2-c2 recipients relative to mOPV2 recipients, and are consistent with prior preclinical and clinical dataEnterovirus C viruses could potentially yield more virulent viruses, but nOPV2 contains modifications aimed at reducing the chances of this happening. Indeed, despite occasional detection of Sabin-1 or −3 by Sabin quadplex rRT-PCR, there was no evidence of nOPV2 recombinant viruses in these samples. Recombination will be evaluated more comprehensively in an analysis of the larger infant cohorts, a separate arm of these same M2 and M5 clinical trials11.The results described here suggest that nOPV2-c1 may increase moderately in fitness and virulence over several weeks replication in the human gut through mutations in the cre5 (U123C/G179A), U459C, VP1-143 and similarly for nOVP2-c2 via U398C and VP1-143. This contrasts with the rapidity of reversion of Sabin-2, whereby highly virulent viruses are selected in all recipients excreting virus longer than 7 days after vaccination. No mechanism for further increases in virulence of nOPV2 is apparent from these results. Recombination with Sabin or A framework Fig.  linking Although there is no direct way to quantitatively extrapolate to reduced risk of paralysis in humans, the available data support the superior genetic and phenotypic stability of shed nOPV2 compared to shed Sabin-2. Furthermore, the data suggest that shed nOPV2 does not (and likely cannot) evolve through single site mutation and selection alone to the level of virulence observed for shed Sabin-2 virus. It is, therefore, likely that the lower/limited evolution of nOPV2 could translate to a lower risk of VAPP relative to mOPV2. Similarly, there is also reduced likelihood of nOPV2 evolving to virulent cVDPV strains following circulation in under-immunized populations.20. As a result of the safety, immunogenicity and promising phenotypic stability of these viruses, Emergency Use Listing (EUL) has recently been granted by WHO for nOPV2-c1 (https://www.who.int/news/item/13-11-2020-first-ever-vaccine-listed-under-who-emergency-use). Initial use of this vaccine under EUL has begun and enhanced pharmacovigilance and environmental surveillance have been implemented to further evaluate this strain under real-world conditions.Vaccination with nOPV2 in outbreak settings features prominently in the Global Polio Eradication Initiative’s new strategy to stop further spread of cVDPV212. Briefly, nOPV2-c1 also includes relocation and modification of the cis-acting replication element (cre) to the 5’ end of the genome, inactivation of cre element in the 2C genomic region by introducing silent mutations, and modifications of the 3D polymerase protein to increase fidelity and reduce recombination13. In addition to the domain V modification, nOPV2-c2 includes synonymous codon deoptimization of the capsid (P1) region, with approximately 40% of the available sites modified through incorporation of CpG dinucleotides in the second and third positions of the codons14.The nOPV2 vaccine candidates are attenuated serotype 2 polioviruses derived from a modified Sabin type-2 infectious cDNA clone. The two candidates  both include the previously described S15 genetic stabilization of domain V in the 5’ UTRProduction lots of nOPV2-c1 and nOPV2-c2 were manufactured and released by P.T. Bio Farma .5 CCID50, Polio Sabin Mono Two, GlaxoSmithKline Biologicals, Belgium) was administered to 50 healthy 1-to-5-year-old Panamanian children who had previously received trivalent OPV (tOPV) or IPV vaccinations21. The protocol was approved by the Ethical Review Committee of the Hospital del Niño 144 “Dr. José Renán Esquivel”. Written consent was obtained from parents or legal guardian(s) at enrollment per country regulations. Trial participants were randomized to receive either one or two doses of mOPV2, with the second dose occurring 4 weeks after the first. Stool samples were collected daily until day 10 following each vaccination, and then on days 14, 21, and 28.In advance of the global type 2 oral poliovirus vaccine cessation in 2016, a Phase 4 trial  was conducted in which mOPV2 .Daily stool samples were collected and periodically shipped to the Centers for Disease Control and Prevention , where they were stored at −20 °C until analysis. Type-2 poliovirus genomes were detected using a Sabin multiplex real-time RT-PCR (rRT-PCR) assay of total nucleic acid extracted from stool suspensions 50 per well) and incubated for three days at 33 °C, 5% CO2. Following two freeze-thaw cycles, cell lysates from the four wells were pooled, and virus isolates were harvested following removal of cell debris by centrifugation at 3000 x g for 10 min. The EES stool suspensions and virus isolates were assigned unique identifiers and shipped on dry ice to Viroclinics Biosciences B.V.  and stored frozen at −80 °C until analysis.Stool suspensions  of the EES were prepared for next-generation sequencing (NGS). To achieve the required concentration of virus for mouse neurovirulence assays, virus was amplified in HEp-2C cells  maintained in EMEM supplemented with 2% fetal bovine serum . Confluent monolayers of HEp-2C cells in 24-well cell culture cluster plates (Costar) were inoculated with a 50 µL aliquot of EES suspension  of nOPV2-c1 or nOPV2-c2. The protocol was approved by the Ethical Review Committee of the Hospital del Niño 144 “Dr. José Renán Esquivel”. Written consent was obtained from parents or legal guardian(s) at enrollment per country regulations.In the phase 2 trial , 49 or 51 healthy 1-to-5-year-old Panamanian participants primarily having received sequential schedules of bOPV and IPV were each orally administered at least one dose  to assess the neurovirulence of shed virus from M2 and M5 clinical trials. The titers of Sabin OPV2 WHO international standard 15/296 (NIBSC) and virus isolates (from EES) were determined by CCID50 assay23 in Hep-2C cells at Viroclinics Biosciences B.V. prior to conducting the mTgmNVT. For each EES, ten 6-to-8-week-old Tg-PVR21 mice (CLEA/Japan) (5 of each sex) were randomized to receive intraspinal inoculations of 104 CCID50 amplified virus (from EES) or control virus in 5 µl. Controls included 20 mice inoculated with Sabin OPV2 virus SO + 2/II at the 5.0 and 6.0 log10 CCID50 dose levels, as well as a sample of shed Sabin-2 virus collected day 7 post-mOPV2-challenge in a prior clinical trial25. Back-titrations of the diluted samples confirmed that titers were within 0.5 log10 CCID50 of the nominal dose. Inoculated mice were monitored for paralysis over a 14-day period per established protocol24.The WHO poliovirus receptor (PVR) transgenic mouse neurovirulence test (TgmNVT) used to release vaccine50) was determined. For these tests, groups of 10 Tg-PVR21 mice were inoculated with each dose and PD50 calculated using logistic regression.When paralysis rates were ≥40%, the paralytic dose for 50% of mice  and according to the DIRECTIVE 2010/63/EU of The European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes under project license 2770020171404 issued by the central authorizing body . Ethical approval for the study was obtained in working protocols 1-3 from the animal ethics committee (Instantie voor Dierenwelzijn) under the above project license.50 values were obtained by inverting the model, with asymptotic confidence intervals computed via the delta method.A binomial logistic generalized linear regression mixed model with mouse gender as a factor and subject (EES)-specific random effects was fitted with SAS version 9.4 to mouse paralysis data for each vaccine group separately, yielding estimates of paralysis rate. For comparison of paralysis rate of shed virus from M5 nOPV2-c1 and nOPV2-c2 participants versus M2 mOPV2, two additional similar models were fit which also included a term to estimate the odds ratio (OR) of paralysis for the virus shed from each candidate relative to shed mOPV2. This estimate is referred to as the adjusted odds ratio (aOR), as it is adjusted for mouse gender imbalance owing to mice excluded from evaluation due to reasons unrelated to the inoculum. Estimated PD27. The method was qualified and executed at Viroclinics Biosciences B.V. In brief, viral RNA was isolated from 140 µl amplified virus stock or stool suspension of each EES using QIAmp Viral RNA mini kit (Qiagen). cDNA preparation and amplification of full-length genome were performed as described elsewhere19. Tagmentation and library preparation by Illumina DNA Prep (M) Tagmentation were performed, followed by 300-cycle paired-end sequencing using MiSeq reagent kit v3 reagents on a MiSeq instrument with analysis software version 1.8.46 (Illumina) to generate FASTQ files. Data analysis was performed using the relevant candidate reference sequence (or to Sabin-2 reference sequence); Genbank accession number AY184220) and a proprietary algorithm at Viroclinics comprised of trimming , mapping , and SNP (single nucleotide polymorphism) calling where multinucleotide polymorphisms and COMPLEXES are split into multiple SNPs using the vcfallelicprimitives utility from vcflib library . Polymorphisms (present at ≥1%) were reported as SNPs with the impact on amino acid coding of the SNP noted per SNP. Re-analysis of select FASTQ files was conducted in Geneious Prime 2020.0.5 (Biomatters) to assess the possible co-location of VP1-143, VP1-N171D and VP1-E295K in nOPV2-c1 shed virus samples16. In brief, for these analyses, reads were merged and mapped against reference sequences from nucleotides 2969-3054 for 143/171 or 3052-3426 for 171/295, requiring the mapped reads to cover the full reference.NGS and data analysis were performed on viral RNA isolated from both cell-culture-amplified virus and from 10% stool suspensions of the EES of each participant, using a previously-described methodThe criteria for reporting in Supplementary Tables Certain EES only had one or two evaluable components (replicates). For EES where both stool aliquots and culture-amplified virus isolate were evaluable, polymorphisms in known attenuation sites and modified regions of the strain present in at least two of the three replicates are indicated in the Supplementary Tables All SNPs reported have Quality scores (Q scores) ≥ 30 unless otherwise indicated in the Supplementary Tables.Mutations associated with amino acid changes present in other regions (outside of key attenuating and modified regions noted above) are provided in the Supplementary Tables Further information on research design is available in the Supplementary InformationREPORTING SUMMARY


Euproctis pseudoconspersa is a major pest of tea plants, and also causes a skin rash on workers in tea plantations. Research on virus could provide fundamental insights for classification, genetic diversity, evolution, and host–virus interaction mechanisms. Here, we identified a novel RNA virus, Euproctis pseudoconspersa bunyavirus (Phenuiviridae), and found that it is widely distributed in field populations of E. pseudoconspersa. The replication of virus in E. pseudoconspersa was indicated by Tag-PCR. These results contribute to the classification of bunyaviruses and provide insight into the diversity of commensal E. pseudoconspersa bunyavirus and the host. Euproctis pseudoconspersa (Strand) (Lepidoptera: Lymantriidae), is one of the major insect pests of tea plants. The larva of this insect pest can also cause allergic reaction on human skin , medium (M), and small (S). Thus, we tentatively named this novel virus as Euproctis pseudoconspersa bunyavirus (EpBYV). Detection of EpBYV by RT-PCR and Sanger-sequencing revealed that it is widely distributed among various field collected populations. In addition, the replication of this virus in tea tussock moths was implied by tag-PCR. These results suggested high level of prevalence of EpBYV in wild population and asymptomatic in the hosts. The actual effect of EpBYV to this host still requires further investigation.In this study, a virus-related sequence was discovered in a field population of Tea tussock moths were collected from tea orchards in July 2017 in Qingyuan district of Guangdong province, China . Total RRaw reads from the RNA-seq library were analyzed by CLC Genomics Workbench 9.5 . After adaptor trimming and filtering of low-quality reads, de novo assembly of clean reads was conducted using Trinity software (RNA). The assembled contigs were subjected to BLASTx and BLASTn searches of viral sequences in GenBank databases. Viral ORFs were predicted using the online tool Gene Finding in Viral Genomes. Conserved and functional domains were identified by searches at the Conserved Domain Database in NCBI and by using the SMART tool. The phylogenetic tree was constructed using the RNA-dependent RNA polymerase (RdRP) amino acid sequence. The tree was constructed with 1,000 bootstrap replicates with the Neighbor-joining method in MEGA 6.0 . The smaE. pseudoconspersa in the main tea cultivation areas of China , 23.9% with Anopheles triannulatus orthophasmavirus (family Phasmaviridae), and 23.5% with Mothra virus (family Phenuiviridae). The S segment contained one ORF (nt 308–1387) encoding a 359-aa protein with conserved domains typical of proteins in the Tenui_N superfamily (NCBI-MN752231). This protein, which was identified as a nucleocapsid protein, showed similarities to nucleocapsid proteins of the genera Tenuivirus (family Phenuiviridae) and Phlebovirus (family Phenuiviridae) with E-value of 5.2 e-10, but differed from those of other members of the Bunyavirales . A bunyavirus in lepidopteran insects has also been reported, namely Hubei lepidoptera virus 1 (NCBI-LCM141331)  was identified from the constructed RNA-seq library. This sequence contained three segments . In the L segment, the ORF from nucleotide position 269 nt to 6940 nt encoded an RdRp (NCBI-MN752229). In the M segment, the nt sequence 190–242 encoded a protein of 744 aa that was annotated as a glycoprotein precursor (NCBI-MN752230). This protein showed identities of 24.6% with avirales . In the M141331) .E. pseudoconspersa, since this could be determined by viral infection stage, viral titters, host immunity hemostasis, and so on, thus further studies are required to analyze the immune response of the hosts in infection of EpBYV.We also explored the viral-derived small RNAs from EpBYV with parallel small RNA sequencing. A library with a 10 M dataset of small RNAs was obtained, but none of these small RNAs were mapped to the genome of EpBYV. In our previous study, we identified a signal of a 22-nt virus-derived small RNA, suggesting that host RNAi is involved in the defense response to bunyavirus infection in aphids. We postulate that the infection of EpBYV may not necessarily induce the response of RNAi as antiviral immunity in E. pseudoconspersa, and the high infection rate of this virus pointed out an existed interaction between the virus and the host. Secondly, by using the above viral positive samples, we detected the viral replication strand by Tag-PCR. The results showed that 4 out of 12 samples presented the replication strand of EpBYV, which were Wanzhou-2018 (sample 3), Anqing-2019 (samples 1 and 3) and Wuxi-2019 (sample 1), respectively , followed by Yongchuan-2017 (95.0%), Yongchuan-2018 (95.0%), Xinyang-2019 (95.0%), Anqing-2019 (85%), Wuxi-2018 (80%), Xingyang-2018 (70%), Wuxi-2019 (55%), Yongchuan-2019 (45.0%), and Guilin-2019 (20%) . The seqectively . Taken t


Circovirus of the family Circoviridae, and it has been associated with porcine circovirus (associated) disease (PCVD or PCVAD) in pigs. PCVAD is the generic term for a series of disease syndromes that have caused economic losses to the pig industry worldwide. Since the discovery of PCV2 in the late 1990s, the virus has continued to evolve, and novel genotypes have continued to appear. Moreover, there has been recombination between different genotypes of PCV2. This review attempts to illustrate some progress concerning PCV2 in genome rearrangement and genomic recombination with non-PCV2-related nucleic acids, particularly focusing on the porcine circovirus-like virus P1 formed by the recombination of PCV2. The presence of rearranged PCV2 genomes can be demonstrated both in vivo and in vitro, and these subviral molecules ranged from 358 to 1,136 bp. Depending on whether it has the ability to encode a protein, the agents formed by PCV2 recombination can be divided into two categories: porcine circovirus-like viruses and porcine circovirus-like mini agents. We mainly discuss the porcine circovirus-like virus P1 regarding genomic characterization, etiology, epidemiology, and pathogenesis. Further research needs to be conducted on the pathogenicity of other porcine circovirus-like viruses and porcine circovirus-like mini agents and the effects of their interactions with PCV2, especially for the porcine circovirus-like mini agents that do not have protein-coding functions in the genome.Porcine circovirus type 2 (PCV2) belongs to the genus  Circoviridae family members have short, circular, single-stranded DNA genomes 1.7–2.0 kb length. The family Circoviridae is divided into two genera, Cyclovirus and Circovirus, with the most studied being porcine circovirus (PCV) and beak and feather disease viruses. The genus Circovirus also includes members of circoviruses that infect a variety of animals, such as avian species , fishes , mammals , and arthropods   .Circoviridae family are considered to be closely related to the members of Nanoviridae and Geminiviridae that infect plants. They all share a stem-loop structure as the origin of viral replication (Ori). Gibbs and Weiller analyzed the origin and evolution of circoviruses through bioinformatic analysis based on Rep protein amino acid sequences of Circovirus and Nanoviridae. The N-terminal amino acid sequences of nanovirus and circovirus Rep were similar, while the C-terminal amino acid sequence of PCV Rep was closely related to the RNA-binding protein (protein 2C) of vertebrate calicivirus. Therefore, the authors hypothesized that PCV originated from the recombination of plant nanoviruses and vertebrate caliciviruses. Since caliciviruses have RNA genomes, retroviruses or retrotransposons may have been involved in the recombination event  in 1974, and it is generally believed that PCV1 is not pathogenic to pigs –15. PCV2The genome of PCVs is very small. The genomes of PCV1 and PCV2 comprise about 1,700 nt, and there are mainly two open reading frames (ORFs). ORF1 is located on the positive strand of the genome and encodes replicases related to viral replication, including rep and rep'. Rep is transcribed from the complete ORF1 and consists of 312 amino acid residues, while Rep' is a spliced transcript with 168 residues . ORF2 is−3 substitutions/site/year , bootstrap support values higher than 70%, and at least 15 available sequences  does not classify viruses below the species level, PCV2 can still be classified into different genotypes. Hamel et al. amplified 438 bp fragments containing each part of ORF1 and ORF2 of PCV2 by PCR, and the fragments were then digested with restriction endonucleases ectively . Wen et ectively . In 2018equences . The firequences , 34. Theequences . Moreoveequences –40. In tCircoviridae and now belonging to the family Anelloviridae, appeared in cell culture and passage in vitro, suggesting that they may play a role in the pathogenesis of TT virus  splicing donor sites are characterized by two identical nucleotides such as AA, TT, GG, or CC  viruses, a type of sub-genome or deletion mutants produced by complete virus replication, and most are replication-deficient. In the traditional sense, the genome of a defective virus is smaller than that of a complete virus; therefore, it can replicate more rapidly when it is co-infected with the complete virus, thereby interfering with the replication of the complete virus. There are DI viruses that can aggravate diseases caused by certain viruses, for example, members of the iviridae .The agents formed by the recombination of PCV2 can be divided into two categories: PCV-like viruses and PCV-like mini agents. The genome of the former has the ability to encode proteins, while the latter does not. Like PCV2, they also have a circular DNA genome. In addition to the high homology of most of the nucleotide sequences to PCV2, their genomes also contain small exogenous nucleotide sequences. Wen et al. have found four PCV-like viruses in pigs, tentatively named P1, P2, ZJ-R (P3), and P4. The full lengths of the viral genomes were 648, 993, 694, and 710 nucleotides, respectively. The lengths of the exogenous nucleotide fragments in the P1, P2, P3, and P4 viral genomes are 16, 281, 180, and 9 nt, respectively . It is sThe current classification of PCVs can be summarized in Since P1 is the first known PCV2 recombinant virus, and the clinical P1 detection rate was comparably higher than that of other PCV-like viruses, we carried out a more systematic study of the P1 virus.The P1 genome is a single-stranded circular DNA and is the animal virus with the smallest currently known genome. Except for 16 nucleotides that are presumed to be derived from porcine retroviruses, the remaining 632 nucleotides are highly homologous with PCV2 ORF2.Although its genome is relatively small, eight ORFs have been verified by PCR, RACE, and Northern blotting. ORF1, ORF2, ORF4, and ORF5 are located on the negative strand of the P1 virus genome, and the positions are 151–493, 492–579, 279–327, and 1–52, respectively. ORF3, ORF6, ORF7, and ORF8 are located on the positive strand of the genome, and the positions are 494–599, 263–293, 285–336, and 374–419, respectively .P1 ORF1 is the largest ORF, encoding a capsid protein of 114 amino acids with a protein size of ~12.5 kDa. Its N-terminal amino acid sequence is highly homologous to the PCV2 capsid protein sequence, while the C-terminal amino acid sequence has no homology with PCV2 capsid protein due to the deletion of one nucleotide corresponding to PCV2. The ORF1 of the original P1 strain encodes 114 amino acids, and a mutant strain appeared in 2017 with ORF1 encoding 122 amino acids . In 2018Using the constructed double-copy tandem molecular cloning of whole P1 genome and DNA site-directed mutagenesis technology, the P1 molecular clones with each ORF deletion were transfected into PK15 cells, and then the total RNA was extracted 12 h later for gene chip analysis. The results showed that P1 ORF1 was involved in the biological processes of defense response to virus, signal transduction, regulation of Rab GTPase activity, and lipid metabolic process; ORF1 was involved in diverse molecular functions of protein phosphatase inhibitor activity, phosphatidylinositol phospholipase C activity, phosphoric diester hydrolase activity, and Rab GTPase activator activity, and in pathways of digestive gland secretion  and neural development , 61.P1 ORF2 was involved in the biological processes of positive regulation of cell growth, proliferation and migration, positive regulation of leukocyte chemotaxis, and defense response to virus; in molecular functions, ORF2 was involved in insulin-like growth factor binding and chemokine activity, and ORF2 was involved in pathways of the cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway, toll-like receptor signal pathway, chemokine signal pathway, and cytokine and cytokine-cytokine receptor interactions .The biological processes, molecular functions, and related pathways involved in P1, ORF3, and ORF5 are basically similar to those of ORF2 .P1 ORF8 is mainly involved in biological processes such as purine nucleotide biosynthetic process, amino acid transport, amino acid transmembrane transport, and defense response to virus and in molecular functions such as AMP-lyase activity and amino acid transmembrane transporter activity .in vivo, PCV-like viruses, and other PCV-like mini agents can be detected in the sera of PMWS cases. In addition, PCV-like mini agents (PCVL258 and PCVL264) can also be detected in the sera of dogs and cats with respiratory diseases or enteric diseases (Both rearrangement molecules and recombinant viruses of PCV2 have been found in pigs. Except for the two PCV-like mini agents (PCVL201 and PCVL347) from the sera of clinically healthy cattle , all PCVdiseases . HoweverThe reports of P1 virus infection are currently only from China. P1 infection was first detected by PCR in 248 pigs from six pig farms in Jiangsu Province, China. The results showed that three farms were P1-positive, with 19% infection rate. About 30% of the P1 antibody positives in pigs in Jiangsu, Zhejiang, Shandong, and Shanghai were detected by the indirect ELISA established by the prokaryotic expression of part of the P1 capsid protein, and the P1 infection rate of pigs with PMWS was significantly higher than that of pigs without PMWS , 63. P1 PK15 cells were transfected with the double-copy tandem molecular clone of the P1 virus genome, and the inclusion bodies were found in the cytoplasm and nucleus of cells by electron microscopy. In comparisons, there was a large number of cytoplasmic inclusion bodies that were basically round, with a diameter of about 0.1–0.3 μm. There were two types of nuclear inclusion bodies: one was small and round with a diameter of about 0.1 μm and high electron density; the other was slightly hexagonal with a size of about 0.4–0.8 μm .The pigs inoculated with the P1 infectious clone through inguinal lymph nodes began to develop viremia in varying degrees at day 14. Some pigs showed similar clinical symptoms as PMWS characterized by weight loss, pale skin, and diarrhea during the experiment . After 3BALB/c mice were artificially infected with P1 rescue virus and divided into a single-challenge group and a three-challenge group . The results showed that on the 14th and 21st days after challenge, the weight gain rate of the mice in the three-challenge group was significantly lower than that in the control group. ELISA antibodies were detected throughout the 35- day experimental period. Viral nucleic acids were detected by PCR in the heart, liver, spleen, lung, bladder, testis, brain, thymus, and pancreas. Viral antigens were detected in the heart, liver, spleen, lung, testis, and thymus using immunohistochemistry. Histopathological changes of mice infected with P1 virus showed interstitial pneumonia, including perivascular connective tissue edema, mild inflammatory cell infiltration, alveolar wall thickening, myocardial necrosis, and Purkinje nucleolysis .Congenital tremor (CT) in piglets is a sporadic disease characterized by paroxysmal muscle spasms. It mostly occurs in newborn piglets without seasonality. The disease was first reported in western Canada in 1992, and has since occurred in many countries. The diseased piglets are born characterized by systemic tremors. Most suffer from severe malnutrition or starvation due to lack of colostrum or colostrum deficiency. In severe cases, the mortality is as high as 100%. In general, there are no obvious changes observed in the central nervous system, peripheral nervous system, or skeletal muscle of the affected piglets through gross anatomical examination. However, different degrees of hypomyelination and mild vacuolization of the central nervous system are typical histological features , 69.Previous studies have shown that CT may be caused by a classical swine fever virus named “Linda virus,” an atypical porcine pestivirus (APPV), an astrovirus, or a porcine teschovirus –74. Wen Besides PCV-like virus P4, PCV-like virus P1 was also detected in the fetal lung tissues of unexplained abortions in pig farms in Jiangsu and Anhui provinces in 2017; these tested negative for classical swine fever virus (CSFV), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), pseudorabies virus (PRV), and PCV2 by PCR or RT-PCR .Syndromes and diseases associated with P1 virus are summarized in Compared with the control group, there were five indicators that displayed no differences in the peripheral blood erythrocyte parameters of pigs infected with PCV-like virus P1: red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpusular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC). While the mean corpuscular hemoglobin (MCH) decreased significantly at 17 days after inoculation, the red blood cell volume distribution width (RDW) increased significantly at 31 days. There was no significant difference in the mean platelet volume (MPV), but the platelet count (PLT), and plateletocrit (PCT) decreased significantly at 8 days after inoculation. The platelet volume distribution width (PDW) decreased significantly at 17 days, suggesting that P1 can lead to small-cell hypochromic anemia characterized by an abnormally low MCH in the red blood cells. Significantly more apoptosis-positive cells at days 21 and 35 after inoculation were detected in the immune organs and tissues  by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assays –78.In the first week after challenge with P1 virus, the MPV of Balb/c mice significantly increased compared with that of the control group. Similarly, in the fifth week after challenge, the MCH and MCHC were significantly lower than those in the control group .The expression of TLRs mRNA was down-regulated at 3 dpi, except for TLR 2 and TLR 10. Since then, TLRs mRNA expression levels of different phases of infection groups were basically in recovery trends. The expression of TLR2 mRNA was significantly increased on the 24th and 40th days after infection, and the expression of TLR9 mRNA on the 31st and 40th days after infection also showed an up-regulated trend. The results suggested that the inflammatory response mediated by TLR2 and TLR9 may play an important role in P1 recognition and its pathogenesis .The mRNA expression levels of cytokines IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, and IFN-γwere relatively different after P1 infection. The mRNA of IL-4 on the 24th day after infection, IL-8 on the 24th and 40th days, and IL-12p40 on the 40th day were significantly up-regulated; IL-6 mRNA was up-regulated during the test period, while IL-10 mRNA was significantly down-regulated on the 3rd and 31st days after infection. The changes in these cytokines can preliminarily clarify that P1 infection may be characterized by excessive inflammatory response, leading to anemia .On the third day after P1 infection, the expression of CD80 mRNA in porcine alveolar macrophages showed a down-regulated trend, and the expression levels of CD40, CD74, and SLA-DR were significantly down-regulated. The expression levels of SLA-DR and SLA-DM on the 8th day and SLA-DM on the 17th day showed a down-regulated trend. The expression of CD86 mRNA showed an up-regulated trend on the 24th day after infection. The expression of CD74 mRNA was significantly down-regulated at 40 dpi. The results revealed that PCV-like virus P1 infection had a significant inhibitory impact on MHC class II processing and presentation of antigens of porcine alveolar macrophages, affecting the immune response .The above-mentioned protein molecules with significant changes at different times after P1 virus infection are summarized in in vitro, the level of MMP-2 mRNA decreased significantly; the level of c-mys mRNA decreased significantly at 24 h. The level of β-catenin protein was down-regulated; it was prevented from entering the nucleus, and TCF/LEF promoter activity was eliminated. The results showed that P1 virus infection could inhibit the Wnt pathway activity both in vivo and in vitro .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Vaccines are considered the path out of the COVID-19 pandemic. The government of Kenya is implementing a phased strategy to vaccinate the Kenyan population, initially targeting populations at high risk of severe disease and infection. We estimated the financial and economic unit costs of procuring and delivering the COVID-19 vaccine in Kenya across various vaccination strategies.n = 12) were done to inform the activities, assumptions and the resources required. Unit prices were derived from document reviews or from market prices. Both financial and economic vaccine procurement costs per person vaccinated with 2-doses, and the vaccine delivery costs per person vaccinated with 2-doses were estimated and reported in 2021USD.We used an activity-based costing approach to estimate the incremental costs of COVID-19 vaccine delivery, from a health systems perspective. Document reviews and key informant interviews and advocacy, communication and social mobilization (29–35%) respectively.This analysis presents cost estimates that can be used to inform local policy and may further inform parameters used in cost-effectiveness models. The results could potentially be adapted and adjusted to country-specific assumptions to enhance applicability in similar low-and middle-income settings. As of March 04 2022, there have been 323,025 confirmed cases and 5640 reported deaths in Kenya [COVID-19 cases and deaths have been on the rise globally and locally since the initial case was reported in Kenya on 13Kenya’s COVID-19 vaccine deployment plan targets to vaccinate 100% of its adult population by December 2022 while prioritizing those who are at an increased risk of severe disease and infection . The couIn Kenya, prior to the international procurement of vaccines, authorization from the Pharmacy and Poison’s board (PPB), the country’s medicine regulatory agency, should be secured. Subsequently in-country vaccine delivery is carried out by the National Vaccine Immunization Program (NVIP), starting with supply chain and logistics, where vaccines and related supplies are distributed from the national depots through the 8 regional depots and eventually to the healthcare facilities . VaccineEvidence on the costs of delivering COVID-19 vaccines are useful for several reasons. First, the financial costs of delivery are useful inputs in decisions and planning about resource mobilization for the vaccine programme. Second the financial costs of delivery provide inputs for assessments of budget impact and affordability of vaccination deployment scenarios, and hence can inform vaccine priority setting decisions. Third, economic costs of vaccine delivery are useful for parameterizing vaccine cost-effectiveness models that will provide evidence on the value for money of different vaccination strategies, and threshold prices that can guide price negotiations to improve the efficiency of procurement. This study aims to estimate the financial and economic unit costs of procuring and delivering the COVID-19 vaccine in Kenya across various vaccination strategies.Kenya is a lower middle-income country with a Gross Domestic Product (GDP) per capita of $1,838.21 . The totThe government of Kenya has a phased approach of vaccine deployment that prioritises the population receiving the vaccine based on their vulnerability, vaccine availability and the health system capacity . Phase 1Vaccination was planned to be administered in identified COVID-19 vaccine provider sites from public and private sectors, as well as progressively including targeted outreaches, and eventually campaigns . These vThis study employed a health-system’s perspective. An activity-based costing approach was used to determine the incremental costs of the introduction of COVID-19 vaccines in Kenya, in line with guidelines for estimating the costs of new vaccines introduction , 11. TheFour different vaccination coverage levels were considered; 30%, 50%, 70%, and 100% of adult Kenyan population receiving two-doses of COVID-19 vaccine at the health facilities, within a one-year period. The selected vaccination scenarios reflect the evolution of Kenya’s COVID-19 vaccine deployment strategy, offered largely in health facilities and in some cases outreaches. The lowest coverage scenario (30%) represents the initial coverage target of Kenya’s COVID-19 deployment plan at a time when the country was less optimistic about the availability of vaccines. Given the unpredictability of vaccine supply, we have assumed that this low coverage scenario might still be reflective of what may happen in practice, despite subsequent revisions of the vaccination strategy to revise the coverage target upwards. The 50%, 70% and 100% coverage scenarios reflect targets in subsequent iterations of the deployment plan.The cost analysis made some assumptions shown in Table Cost of vaccine procurement per person vaccinated with two-dosesCost of vaccine-related supplies procurement per person vaccinated with two-dosesCost of vaccine delivery per person vaccinated with two-dosesWe estimated the following unit costs Fig. :Cost of Procurement costs refer to the purchasing costs of the COVID-19 vaccine and its related supplies, accounting for the importation and clearance costs into the country. The vaccine procurement cost per person vaccinated with two-doses and the cost of vaccine related supplies procurement per person vaccinated with two-doses was estimated by dividing the total costs of vaccine procurement and the total costs of vaccine-related supplies procurement respectively by the total number of people targeted for vaccination.Delivery costs refer to the costs of activities required to supply and distribute the vaccine from the national level to the health facilities, as well as activities that ensure the safe administration and uptake of the vaccine. Cost of vaccine delivery per person vaccinated with two-doses was estimated by obtaining the total costs of 1) vaccine supply chain 2) vaccine safety monitoring and adverse events following immunization (AEFI) management 3) training 4) advocacy, communication and social mobilization 5) data management, monitoring and supervision, and 6) vaccine administration and dividing this by the total number of people targeted for vaccination.i)Vaccine and related supplies procurement.This included the costs to procure the vaccine and its related supplies. Vaccine procurement base costs was estimated at US$7 per dose, which is the country’s procurement cost from the COVAX facility. Additionally, freight and insurance fees, handling costs, clearance charges, import declaration fees, and railway development levies were added as a percentage of the base cost of vaccine and related supplies costs. The number of doses to be procured was estimated based on the number of doses to be given and adjusted for the wastage rates.ii)Vaccine supply chain costsVaccine supply chain costs included the costs of distribution of the vaccines and related supplies from the national depots, through the regional depots and eventually to the healthcare facilities. This included capital costs such as the procurement of cold chain equipment, waste management and control equipment, vehicles for last mile distribution, and renovations of county vaccine stores. Recurrent costs included vehicle maintenance costs, waste management and infection control supplies, warehouse costs, transport costs of vaccines and dry supplies, and cold chain preventive maintenance visits.The number of resources, frequency of distribution were estimated at a national level and the total amount of resources required estimated using ingredients costing of the different inputs such as supplies, utilities of the warehouses, fuel, per diems of the drivers and other staff involved in the distribution process, and staff salary costs. The capital investments were valued as a shared input with other vaccines in the Expanded Programme on Immunization (EPI). Therefore, in order to account only for the incremental costs, apportioning of the capital costs was done by allocating a fraction of distribution costs to COVID-19 vaccine based on all vaccine doses given in Kenya in one year.iii)Vaccine safety monitoring & AEFI managementVaccine safety monitoring and AEFI management included sub-activities to support AEFI field investigation, sensitization meetings on vaccine safety and risk management, pharmacovigilance sensitizations and supervision support, quality assurance for supply and vaccine safety, printing of national AEFI guidelines and forms and vaccine safety study. Costs of these sub-activities included conference packages, printing, fuel, airtime, per diem allowances for travel and lunch, and salaries.iv)TrainingThis included the initial training of logisticians at a national and county level, initial training of sub-county health management team (SCHMT) supervisors, and the initial training of vaccinators and volunteers. Additionally, review meetings of the vaccination program at a national, county and sub-county level were included. For each of the trainings and review meetings the estimated costs included conference packages, per diems for participants and facilitators, lunch costs, transport costs, accommodation costs, where necessary, and other supplies.v)Advocacy, communication and social mobilizationThis included activities such as national stakeholder forums, national and county advocacy and launches, media advertisements, information, education and communication material. Further, community mobilization activities with opinion leaders, through roadshows, and with public address systems were also included. The costs for the aforementioned activities included conference packages, per diem allowances for all staff involved, fuel, advertisement production, advertisement truck and generator hire, printing, salaries, and other supplies.vi)Data management, monitoring and supervisionAt a national level this included activities to support national coordinators and supervisors, policy level oversight, microplanning, preparedness assessments, financial oversight, monitoring and evaluation strengthening, national level support, coordination and planning. At a county level this included activities to support SCHMT supervision, county health management team supervision and microplanning. Costs associated with monitoring and supervision activities included per diem and allowances, lunch, fuel, monitoring tools, ICT equipment maintenance, District Health Information Software 2 (DHIS2) customization, evaluation costs, stationaries, salaries, and other supplies. It also included the electronic logistics management information system that is used by government to manage stocks and immunization. Specifically, the system development costs, cloud hosting service charges, tablets used in facilities, and the messaging services to clients.vii)Vaccine administration costsThe different activities identified in the delivery and implementation of the COVID-19 vaccine were categorized under 7 key components; 1) Vaccine and related supplies procurement costs 2) Vaccine supply chain costs 3) Vaccine safety monitoring and AEFI management 4) Training 5) Advocacy, communication, and social mobilization 6) Data management, monitoring and supervision 7)Vaccine administration during service delivery. Under each cost component, there were identified sub-activities and inputs that were costed. This is illustrated in Fig. The service delivery activities included costs associated with vaccine administration in health facilities. These costs included the time and resources needed for each vaccination, including the preparation for vaccine administration, actual vaccination, and post-vaccination reporting. The costs included staff salaries, supplies and building space for vaccine administration. In this analysis we assume that 30% adult vaccination coverage will be administered in all the 3,000 facilities designated for Phase 2 vaccination while coverages of 50, 70, and 100% will be administered in all 7877-facilities country wide designated for Phase 3 rollout. Outreach costs were also included and assumed 25% of the identified facilities would conduct at least two outreaches per week in the second half of the year. These outreach costs included salaries, transport, and lunch costs.Resource use data of the identified inputs classified under each sub-activity and cost component were collected through document reviews of national and county level expenditure plans for COVID-19 deployment and structured interviews with purposefully selected national and county level officers involved in the COVID-19 vaccine deployment. Written informed consent was sought before the commencement of each interview. Specifically, we interviewed 4 officers from the National COVID-19 vaccine deployment taskforce, and 8 officers involved in county deployment and administration of COVID-19 vaccines in public health facilities drawn from purposely selected, geographically diverse, counties , Table First, costs were classified as recurrent versus capital costs. Recurrent costs included resources that are expected to be replaced/consumed within one year. These included costs related to vaccine and related supplies, personnel, transport, all supplies, operations and maintenance, meetings, monitoring and supervision activities, advocacy activities, social mobilization activities. Capital costs on the other hand included the value of resources that provide services for more than one year. These included the costs of equipment, vehicles, building space, and initial training, development costs of the electronic logistics management information system, and the related cloud hosting costs. The useful life of vehicles, computers, and the electronic management system was estimated at 5 years, while that of training, large equipment and machinery, and buildings was estimated at 2 years, 10 years, and 30 years respectively. Equipment and vehicles were assumed to be used for other vaccines as well and therefore their annuitized costs were apportioned based on total EPI and COVID-19 vaccines given in one year.Second, costs were also classified as either financial or economic costs. Financial costs only reflect the actual costs incurred by the government of Kenya for COVID-19 vaccines and its delivery. For instance, financial costs excluded the vaccine costs procured by COVAX facility and those donated through bilateral negotiations, salaries of all staff involved, volunteer costs, and development and cloud hosting of the electronic logistics management information system. On the other hand, economic costs reflect the opportunity costs and covers the value of all resources used including those not captured in financial outlays by estimating their value. Therefore, economic costs included all financial costs as well as the value of donated vaccines, volunteer time, and staff salaries. For each cost component, both financial and economic costs are estimated.All costs were valued using purchase prices from the financial records, in few instances where this was not available, the replacement costs from similar items was used. Volunteer costs were valued based on the minimum wage in Kenya for a general labourer . All capA univariate sensitivity analysis was done to determine the robustness of the unit cost estimates with variations in key parameters on the economic costs. First, the base costs were varied to range a minimum of $3 to a maximum of $10 assuming that the government of Kenya procures vaccines from Africa Union at different prices . Second,A total of 8.0 million, 13.4 million, 18.7 million, and 26.8 million adults (above the age of 18) from the Kenyan population are targeted for vaccination under the 30% coverage, 50% coverage, 70% coverage, and 100% coverage vaccination strategies respectively. This is reported in Table The reported total costs reflect 2-vaccine doses per person vaccinated. The total financial procurement costs ranged from $24.55 million to $352.74 million while the economic procurement costs ranged from $140.65 million to $468.84 million for the 30 and 100% coverage levels respectively.The total financial delivery costs ranged from $34.39 million to $87.98 million for the 30% and 100% coverage levels respectively. The total economic delivery costs ranged from $98.18 million to $192.09 million for the 30%and 100% coverage levels respectively.The total financial costs of both procurement and delivery was estimated at $58.94 million, $168.50 million, $278.62 million, and $440.72 million for the 30%, 50%. 70% and 100% coverage levels respectively. Further, the total economic costs of both the procurement and delivery was estimated at $238.83 million, $345.78 million, $474.26 million, and 660.94 million for the 30%, 50%, 70%, and 100% coverage levels respectively. These total incremental costs of procurement and delivery of COVID-19 vaccines are reported in Table Vaccine procurement costs are the largest cost-driver, accounting for 39% to 79% of total financial costs and 58% to 70% of total economic costs across the different vaccine strategies.When all procurement costs are excluded, and only delivery costs are considered; the major financial cost driver include vaccine supply chain activities accounting for 47% to 59% of total financial delivery costs. This is then followed by advocacy, communication and social mobilization activities that are estimated at 18–19% of total financial delivery costs and data management, monitoring &supervision activities that are 12% to 21% of total financial delivery costs. However, for economic delivery costs, the main cost driver is advocacy, communication and social mobilization activities accounting for 29% to 35% of total economic delivery costs. This is then followed by supply chain activities, and vaccine administration activities that account for 16% to 27% and 17% to 27% of total economic delivery costs respectively. Across both financial and economic costs, training activities, and vaccine safety monitoring & AEFI management activities were the least cost drivers for the delivery costs. This is illustrated in Fig. The financial vaccine procurement unit costs to be incurred by the government of Kenya was estimated at $2.89, $8.67, $11.15, and $13.01 per person vaccinated with two-doses for the 30%, 50%, 70% and 100% coverage levels respectively. While the full economic costs of vaccination procurement per person vaccinated with 2 doses was $17.34.The financial costs of vaccine delivery per person vaccinated with two-doses ranged from $3.29 to $4.28 and the economic costs of vaccine delivery per person vaccinated with two-doses ranged from $7.17 to $12.22 in the 100% and 30% coverage levels respectively.The total financial unit costs per person vaccinated with two doses (procurement and delivery costs) ranged from $7.34 to $16.47 and the total economic unit costs per person vaccinated with two doses ranged from $29.73 to $24.68 in the 30% and 100% coverage levels. The unit costs are reported in Table The economic cost of vaccine procurement per person was sensitive to changes in vaccine prices, with a 56% decrease ($7.60) and a 44% increase ($24.94) in the unit cost. The cost of vaccine delivery per person were robust to the changes in the parameters. The economic vaccine delivery per person increased and decreased by less than 10% across all vaccination strategies. This is illustrated in Fig. We estimated the costs of procuring and delivering the COVID-19 vaccine in Kenya under four different vaccination coverage scenarios. This study provides insights into country-specific unit costs and cost drivers of COVID-19 vaccine delivery costs that can be useful for planning and budgeting purposes and contributes to evidence of costing studies of COVID-19 vaccine delivery.The largest costs driver was the costs to procure vaccines which contributed 39% to 79% of total financial costs and 58% to 70% of total economic costs across the different vaccination strategies. This is consistent with other vaccine costing studies for other diseases; For instance, a multi-country study done across Ghana, Kenya, and Malawi estimated that the costs of the malaria vaccine were up to 90% of the total financial and economic costs of continuing malaria vaccination after piloting . The senThis study estimates the financial costs of COVID-19 vaccine delivery per person at US$4.28, US$3.75, US$3.55, and US$3.29 for the 30%, 50%, 70% and 100% coverage vaccination strategies. Further, the reported economic costs of COVID-19 vaccine delivery range from US$7.17 to US$12.22 across the vaccination strategies. As expected, the economic costs are consistently higher than the financial costs due to the inclusion of opportunity costs such as salaries, volunteer labour, donated goods, and building space and the development and cloud hosting of the electronic logistics management information system. The estimated unit costs in this study also indicate possible economies of scale in COVID-19 vaccine delivery as the target coverage increases.Although not directly comparable, a costing study in 92 countries estimated the financial COVID-19 delivery costs of US$3.70 per person vaccinated with two doses for a 20% target population . Our estOur findings demonstrated that when considering the incremental financial costs for COVID-19 vaccine delivery, supply chain activities followed by advocacy, communication and social mobilization, and data management, monitoring and supervision were the top three cost drivers. However, for economic costs, vaccine administration costs during service delivery was the third cost driver in addition to supply chain and advocacy, communication and social mobilization. Previous evidence, that focused on total costs reported supply chain costs to be lower than immunization services for EPI vaccines in Kenya . For malThis study’s limitation is that the study relied on expenditure plans rather than actual expenditures. However, this was mitigated by augmenting this data with structured interviews with vaccine deployment implementors. Further, this analysis focussed on a 2-dose vaccine formulation, future analysis should consider vaccine delivery costs per person for 1-dose formulations, as well as vaccine strategies that include boosters. The strength of this paper is that the assumptions, unit prices, and vaccination scenarios used in the cost analysis are country-specific and therefore can be used to inform local policy and may further inform parameters used in cost-effectiveness models.This analysis presents cost estimates and highlights cost drivers of the COVID-19 delivery strategy that targets different coverage levels. This can be used to inform local policy and may further inform parameters used in cost-effectiveness models. The results could potentially be adapted and adjusted to country-specific assumptions to enhance applicability in similar low-and middle-income settings.


Triticum aestivum L.) plants can be co-infected by multiple viruses, including wheat streak mosaic virus (WSMV), Triticum mosaic virus (TriMV), brome mosaic virus (BMV), and barley stripe mosaic virus (BSMV). These viruses belong to four different genera in three different families and are, hence, genetically divergent. However, the impact of potential co-infections with two, three, or all four of them on the viruses themselves, as well as the wheat host, has yet to be examined. This study examined bi-, tri-, and quadripartite interactions among these viruses in wheat for disease development and accumulation of viral genomic RNAs, in comparison with single virus infections. Co-infection of wheat by BMV and BSMV resulted in BMV-like symptoms with a drastic reduction in BSMV genomic RNA copies and coat protein accumulation, suggesting an antagonism-like effect exerted by BMV toward BSMV. However, co-infection of either BMV or BSMV with WSMV or TriMV led to more severe disease than singly infected wheat, but with a decrease or no significant change in titers of interacting viruses in the presence of BMV or BSMV, respectively. These results were in stark contrast with exacerbated disease phenotype accompanied with enhanced virus titers caused by WSMV and TriMV co-infection. Co-infection of wheat by WSMV, TriMV, and BMV or BSMV resulted in enhanced synergistic disease accompanied by increased accumulation of TriMV and BMV but not WSMV or BSMV. Quadripartite interactions in co-infected wheat by all four viruses resulted in very severe disease synergism, leading to the death of the most infected plants, but paradoxically, a drastic reduction in BSMV titer. Our results indicate that interactions among different viruses infecting the same plant host are more complex than previously thought, do not always entail increases in virus titers, and likely involve multiple mechanisms. These findings lay the foundation for additional mechanistic dissections of synergistic interactions among unrelated plant viruses.Field-grown wheat ( Virus–virus interactions of plant viruses can lead to superinfection exclusion (SIE) and synergistic interaction between related and unrelated viruses, respectively . SIE is In mixed infections, the interaction between unrelated viruses can result in disease synergism or neutralism with enhanced and no effect on symptom phenotype, respectively . Mixed iSynergistic interaction is manifested by an increase in virus titer of one or two interacting viruses with an increase in symptom phenotype compared to infections by individual viruses. Mixed infections can cause disease synergism due to an increase in viral replication, viral movement, and interference with host defense mechanisms. Co-infection of tobacco plants by potato virus X (a potexvirus) and a potyvirus  resulted in disease synergism with an increased accumulation of potato virus X but not the potyvirus . In contIn the Great Plains region of North America, viral diseases can cause 3–5% annual yield losses in wheat, with $76 million yield loss in Kansas alone . AgropyrTritimovirus and Poacevirus genera, respectively, in the family Potyviridae (Aceria tosichella Keifer)  . The gen Keifer) . Since t Keifer) , 2014.Bromovirus in the family Bromoviridae with a tripartite single-stranded positive-sense RNA genome of RNA1 (3.2 kb), RNA2 (2.9 kb), and RNA3 (2.1 kb) (Hordeivirus in the family Virgaviridae with a tripartite genome of RNAα (3.8 kb), RNAβ (3.2 kb), and RNAγ (2.8 kb) (Brome mosaic virus is the type member of the genus (2.1 kb) . These t(2.1 kb) . BMV is (2.1 kb) . Co-infe(2.1 kb) . BSMV is(2.8 kb) . Since B(2.8 kb) , there iPreviously, synergistic interaction between WSMV and TriMV, two distinct potyvirid species, was examined in wheat , 2014. Hin vitro transcripts of pSP6-WSMV and pTriMV-R, respectively  (/CI(7aa)  and pTri/CI(7aa)  were inoWheat cv. Arapahoe seedlings were raised in 15 cm-diameter earthen pots filled with pasteurized soil mix consisting of 34% each of clay loam soil and peat moss, and 16% each of sand and vermiculite. Wheat leaves infected with WSMV, TriMV, BMV, or BSMV were ground in a mortar and pestle in 20 mM sodium phosphate buffer, pH 7.0 (inoculation buffer) at 1:10 dilution (1 g tissue in 9 ml inoculation buffer). Ten ml of WSMV crude sap was mixed with 10 ml of inoculation buffer and 10 ml of TriMV, BMV, or BSMV to obtain 1:30 dilution of WSMV + TriMV, WSMV + BMV, and WSMV + BSMV, respectively. Similarly, 10 ml of TriMV crude sap was mixed with 10 ml of inoculation buffer and 10 ml of BMV or BSMV to obtain TriMV + BMV and TriMV + BSMV, respectively. BMV + BSMV inoculum was obtained by mixing 10 ml each of BMV, BSMV, and inoculation buffer. WSMV + TriMV + BMV or BSMV inocula were obtained by mixing 10 ml of WSMV, TriMV, and BSMV or BSMV, respectively. Inoculum consisting of all four viruses was obtained by mixing equal volumes of WSMV, TriMV, BMV, and BSMV to obtain 1:40 dilution. Virus inocula consisting of one, two, and three viruses were used at 1:30 dilution, and inoculum consisting of all four viruses was used at 1:40 dilution.Three carborundum-dusted pots of wheat seedlings (12–16 seedlings per pot with 36–48 seedlings per treatment) at the single leaf stage were mechanically inoculated with inoculation buffer (mock) or with crude sap containing one, two, three, or four viruses. Inoculated wheat pots were kept in a greenhouse at 24–26°C maximum and 20–22°C minimum temperature with ∼16 h daylight/supplemental light. Cultural practices and routine pesticide applications were performed to control wheat curl mites and insects on wheat plants. The mock-inoculated wheat plants remained virus-free through the course of this experiment. Wheat seedlings inoculated with virus and mock were observed daily starting at 6 dpi, and symptoms were recorded at 7, 10, 14, 20, and 30 dpi. Symptoms elicited by viruses were photographed at 10, 20, and 30 dpi. The entire experiment was repeated two and three times to determine viral genomic RNA accumulation and symptom phenotype, respectively.g for 15 min at 4°C. Total RNA was precipitated from 150 μl of aqueous phase by adding an equal volume of isopropanol, and the tubes were stored at room temperature for 10 min. Total RNA was pelleted by centrifugation at 12,000 × g for 10 min at 4°C, and the RNA pellet was washed with 450 μl of 70% ethanol, followed by centrifugation at 12,000 × g for 5 min. The RNA pellet was dried in a speed vac for 20 min and suspended in 150 μl sterile water. Total RNA was quantified using a NanoVue Plus Spectrophotometer  and stored at −80°C.The upper, fully expanded wheat leaves collected at 10, 20, and 30 dpi from the virus- or mock-inoculated wheat plants were used for total RNA extraction. Total RNA was isolated from 200 mg tissue in triplicate from virus-infected and mock-inoculated tissue as described in Total RNA isolated from wheat leaves infected by four individual viruses  or co-infections in all combinations  at 10, 20, and 30 dpi and mock-inoculated wheat leaves were used for first-strand cDNA synthesis. Total RNA (1.0 μg) isolated from virus-infected or mock-inoculated wheat leaves were used for first-strand cDNA synthesis in a 20 μl reaction volume. Each first-strand cDNA reaction consisted of 1X first-strand reaction buffer, 100 μM dNTPs, 2.5 ng/μl random primers , and 8.8 U of avian myeloblastosis virus reverse transcriptase . The cDNA synthesis was carried out at 42°C for 1 h and it was followed by termination at 95°C for 5 min. The synthesized cDNA was stored at −20°C until used for real-time PCR.Absolute quantification of WSMV, TriMV, BMV, and BSMV genomic RNA copies was determined in 25 μl reaction volumes with TaqMan DNA polymerase (Applied Biosystems) in Applied Biosystems 7300 Real-Time PCR system  as described in The standard amplification protocol provided with the real-time PCR system was used at 50°C for 2 min, 95°C for 15 min, followed by 40 cycles at 95°C for 15 s and 58°C for 60 s. All reactions were performed in triplicate with virus-specific primers and probe for the detection of WSMV, TriMV, BMV, or BSMV. Primers (50 nm) and probe (200 nM) specific to plant 18S rRNA (Applied Biosystems) was used as an internal reference gene. 6-FAM was used as a label for oligonucleotide probes used for the detection of WSMV, TriMV, BMV, and BSMV, while the VIC label was used for oligonucleotide probe used for 18S rRNA.Standard curves for absolute quantification of WSMV, TriMV, BMV, and BSMV were prepared from serially diluted agarose gel-purified virus-specific PCR fragments. The number of copies in gel-purified PCR fragments that were used to make standard curves was calculated as described in The Q-Gene software was used to calculate the absolute number of genomic RNA copies of WSMV, TriMV, BMV, and BSMV in single and mixed infections of wheat . The Ct Total proteins from wheat leaves infected with BMV, BSMV, or BMV + BSMV at 10, 14, and 21 dpi were isolated as described in Two pots of wheat cv. Tomahawk seedlings (12–16 seedlings per pot) at the single-leaf stage were inoculated with crude sap of WSMV-RFP, TriMV-GFP, WSMV-RFP + TriMV-GFP, WSMV-RFP + BMV, WSMV-RFP + BSMV, TriMV-GFP + BMV, or TriMV-GFP + BSMV. Virus-inoculated wheat pots were kept in a greenhouse with 16 h daylight/artificial light. The uppermost leaves from 10 different plants were examined under a Stereo Discovery V12 Fluorescence Dissecting Microscope  using a narrow band of filter set of RFP or GFP, and fluorescent images were obtained with the AxioCam MRc5 camera as described in To examine the interaction between BMV and BSMV, wheat seedlings at the single-leaf stage were inoculated with BMV, BSMV, or BMV + BSMV and the symptom development was monitored. BMV elicited severe chlorotic streaks and spots with mild yellowing of leaves at 7 dpi, followed by moderate chlorotic streaks and mosaic symptoms at 10 dpi . Subsequ6, 3.1 × 106, and 5.3 × 106 at 10, 20, and 30 dpi, respectively, in BMV infected wheat  and TriMV (not significantly) compared to individual infections. To further examine whether the presence of BMV or BSMV in co-infected wheat has any effect on the expression of WSMV and TriMV, an RFP-tagged WSMV or GFP-tagged TriMV was used to co-infect wheat with BMV or BSMV. At 14 dpi, expression of RFP was substantially reduced in wheat co-infected by WSMV-RFP + BMV compared to those of WSMV-RFP + TriMV-GFP or WSMV-RFP , suggestSynergistic interactions between two unrelated viruses in plants have been examined extensively for several virus combinations . HoweverMixed infections by unrelated viruses would lead to synergism with enhanced disease or neutralism with no effect on disease compared to infections by individual viruses . In contPreviously, we reported disease synergism in co-infected wheat by WSMV and TriMV with a substantial increase in titers of both interacting viruses , 2019. IIn tripartite interactions, the presence of either BMV or BSMV with WSMV and TriMV elicited enhanced disease synergism compared to co-infection by WSMV and TriMV. Co-infection of wheat by WSMV and TriMV caused enhanced titers of both interacting viruses . HoweverOf all the interactions examined in this study, the quadripartite interactions in wheat co-infected by all four viruses caused the most severe synergism, with severe stunting and death of most plants. These data suggest that the presence of BMV and BSMV with WSMV and TriMV caused additive effects on synergistic interactions that resulted in lethal synergistic disease. Quadripartite interactions in wheat resulted in no significant change in WSMV and TriMV titers, increased BMV titer, and a drastic reduction in BSMV titer. Accumulation of BSMV genomic RNA copies did not change significantly in BSMV co-infected with WSMV and TriMV, but the addition of BMV to BSMV, WSMV, and TriMV resulted in an 8- to 25-fold decrease in BSMV titer. These data further confirm that the presence of BMV in co-infected wheat affected BSMV replication.Antagonistic-like interactions between unrelated BMV and BSMV in co-infected wheat resulted in a 2- to 33-fold decrease in BSMV titer. In synergistic interactions, suppressors of RNA silencing proteins of interacting viruses play an important role in causing the disease synergism . The γb In mixed infections, efficient replication of one of the interacting viruses likely plays an important role in determining the titers of other interacting partner viruses. In singly infected wheat, BMV accumulated at 50-76-, 47-72-, and 2.4-5.1-fold more than WSMV, TriMV, and BSMV, respectively. BSMV accumulated at 15-27-, 14-28-, and 0.2-0.4-fold of WSMV, TriMV, and BMV, respectively, in singly infected wheat. These data further support that the robust replication of tripartite BMV in wheat might have depleted the shared resources for replication of another tripartite virus. Interestingly, BMV repressed BSMV accumulation but not WSMV and TriMV in quadripartite interactions. The robust replication of BMV might have also depleted host resources for moderately replicating WSMV or TriMV in mixed infections in wheat; thus, BMV caused decreased genomic RNA accumulation and gene expression of WSMV and TriMV in bipartite interactions. However, efficient suppressors of RNA silencing of WSMV  and TriMPreviously, we found that during the early stages of synergistic interaction between WSMV and TriMV, prior infection of wheat by TriMV facilitated the accelerated long-distance movement of WSMV . In contThe original contributions presented in the study are included in the article/ST formulated and designed the experiments and wrote the manuscript. ST and JA performed the research. ST, FQ, and JA analyzed the data, edited the manuscript, and approved the submitted version.USDA is an equal opportunity provider and employer. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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