Current state-of-the-art and potential future therapeutic drugs against COVID-19.

The novel coronavirus disease (COVID-19) continues to endanger human health, and its therapeutic drugs are under intensive research and development. Identifying the efficacy and toxicity of drugs in animal models is helpful for further screening of effective medications, which is also a prerequisite for drugs to enter clinical trials. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) invades host cells mainly by the S protein on its surface. After the SARS-CoV-2 RNA genome is injected into the cells, M protein will help assemble and release new viruses. RdRp is crucial for virus replication, assembly, and release of new virus particles. This review analyzes and discusses 26 anti-SARS-CoV-2 drugs based on their mechanism of action, effectiveness and safety in different animal models. We propose five drugs to be the most promising to enter the next stage of clinical trial research, thus providing a reference for future drug development.

CBFß is induced by spring viremia of carp virus and promotes virus replication in zebrafish.

The core binding factor subunit beta (CBFß) is a transcription factor that forms a complex with virial proteins to promote viral infection. In this study, we identified a CBFß homolog from zebrafish (zfCBFß) and characterized the biological activity. The deduced zfCBFß protein was highly similar to orthologs from other species. The zfcbfß gene was constitutively expressed in tissues and was induced in immune tissues after infection with spring viremia carp virus (SVCV) and stimulation with poly(I:C). Interestingly, zfcbfß is not induced by type I interferons. Overexpression of zfcbfß induced tnfα expression but inhibited isg15 expression. Also, overexpression of zfcbfß significantly increased SVCV titer in the EPC cells. Co-immunoprecipitation assay revealed that zfCBFß interacts with SVCV phosphoprotein (SVCVP) and host p53, resulting in the increased stability of zfCBFß. Our results provide evidence that CBFß is targeted by virus to suppress host antiviral response.

Delivery of progeny virus from the infectious clone of cucumber green mottle mosaic virus and quantification of the viral load in different host plants.

Cucumber green mottle mosaic virus (CGMMV, genus Tobamovirus) is a widely occurring tobamovirus in cucurbits. The genome of CGMMV has been used previously for the expression of foreign genes in the plant. High throughput delivery and high viral titer are important requirements of foreign protein expression in plant through virus genome-based vector, in this study, Agrobacterium containing infectious construct of CGMMV was infiltrated through syringe, vacuum and high-speed spray to N. benthamiana, cucumber and bottle gourd leaves. The success rate of systemic infection of CGMMV agro-construct through all three methods was higher (80-100%) in N. benthamiana compared to the cucurbits (40-73.3%). To determine the high-throughput delivery of CGMMV in the plant system, four delivery methods viz. rubbing, syringe infiltration, vacuum infiltration and high-speed spray using the progeny virus derived through CGMMV agro-construct were compared in the three different plant species. Based on the rate of systemic infection and time required to perform delivery by different methods, vacuum infiltration was found most efficient for the high-throughput delivery of CGMMV. The quantification of CGMMV through qPCR revealed that CGMMV load varied considerably in leaf and fruit tissues depending with the time of infection. Immediately after expression of symptoms, a high load of CGMMV (~ 1 µg/100 mg of tissues) was noticed in young leaves of N. benthamiana and cucumber. In bottle gourd leaves, the CGMMV load was far low compared to N. benthamiana and cucumber plants. In the fruit tissues of cucumber and bottle gourd higher virus load was observed in mature fruit but not in immature fruit. The findings of the present study will serve as an important base line information to produce foreign protein through CGMMV genome-vector. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03630-y.

Efficacy and Safety of Ensitrelvir in Patients With Mild-to-Moderate Coronavirus Disease 2019: The Phase 2b Part of a Randomized, Placebo-Controlled, Phase 2/3 Study.

BACKGROUND: This phase 2b part of a randomized phase 2/3 study assessed the efficacy and safety of ensitrelvir for mild-to-moderate coronavirus disease 2019 (COVID-19) during the Omicron epidemic. METHODS: Patients were randomized (1:1:1) to orally receive ensitrelvir fumaric acid 125 mg (375 mg on day 1) or 250 mg (750 mg on day 1) or placebo once daily for 5 days. The co-primary endpoints were the change from baseline in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) titer on day 4 and time-weighted average change from baseline up to 120 hours in the total score of predefined 12 COVID-19 symptoms. Safety was assessed through adverse events. RESULTS: A total of 341 patients (ensitrelvir 125-mg group: 114; ensitrelvir 250-mg group: 116; and placebo group: 111; male: 53.5-64.9%; mean age: 35.3-37.3 years) were included in the efficacy analyses. The change from baseline in SARS-CoV-2 titer on day 4 was significantly greater with both ensitrelvir doses than with placebo (differences from placebo: -0.41 log10 50% tissue-culture infectious dose/mL; P < .0001 for both). The total score of the 12 COVID-19 symptoms did not show a significant difference between the ensitrelvir groups and placebo group. The time-weighted average change from baseline up to 120 hours was significantly greater with ensitrelvir versus placebo in several subtotal scores, including acute symptoms and respiratory symptoms. Most adverse events were mild in severity. CONCLUSIONS: Ensitrelvir treatment demonstrated a favorable antiviral efficacy and potential clinical benefit with an acceptable safety profile. CLINICAL TRIALS REGISTRATION: Japan Registry of Clinical Trials: jRCT2031210350 (https://jrct.niph.go.jp/en-latest-detail/jRCT2031210350).

Brazilian Populations of Aedes aegypti Resistant to Pyriproxyfen Exhibit Lower Susceptibility to Infection with Zika Virus.

Zika virus (ZIKV) infection has caused devastating consequences in Brazil as infections were associated with neurological complications in neonates. Aedes aegypti is the primary vector of ZIKV, and the evolution of insecticide resistance (IR) in this species can compromise control efforts. Although relative levels of phenotypic IR in mosquitoes can change considerably over time, its influence on vector competence for arboviruses is unclear. Pyriproxyfen (PPF)-resistant populations of Ae. aegypti were collected from five municipalities located in Northeast of Brazil, which demonstrated different resistance levels; low (Serrinha, Brumado), moderate (Juazeiro do Norte, Itabuna), and high (Quixadá). Experimental per os infection using ZIKV were performed with individuals from these populations and with an insecticide susceptible strain (Rockefeller) to determine their relative vector competence for ZIKV. Although all populations were competent to transmit ZIKV, mosquitoes derived from populations with moderate to high levels of IR exhibited similar or lower susceptibility to ZIKV infection than those from populations with low IR or the susceptible strain. These observations suggest an association between IR and arbovirus infection, which may be attributable to genetic hitchhiking. The use of PPF to control Brazilian Ae. aegypti may be associated with an indirect benefit of reduced susceptibility to infection, but no changes in disseminated infection and transmission of ZIKV among PPF-resistant phenotypes.

A Randomized Phase 2/3 Study of Ensitrelvir, a Novel Oral SARS-CoV-2 3C-Like Protease Inhibitor, in Japanese Patients with Mild-to-Moderate COVID-19 or Asymptomatic SARS-CoV-2 Infection: Results of the Phase 2a Part.

This multicenter, double-blind, phase 2a part of a phase 2/3 study assessed the efficacy and safety of ensitrelvir, a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like protease inhibitor, in Japanese patients with mild-to-moderate coronavirus disease 2019 (COVID-19) or asymptomatic SARS-CoV-2 infection. Sixty-nine patients were randomized (1:1:1) to orally receive 5-day ensitrelvir fumaric acid (375 mg on day 1 followed by 125 mg daily, or 750 mg on day 1 followed by 250 mg daily) or placebo and followed up until day 28. The primary outcome was the change from baseline in the SARS-CoV-2 viral titer. A total of 16, 14, and 17 patients in the ensitrelvir 125 mg, ensitrelvir 250 mg, and placebo groups, respectively, were included in the intention-to-treat population (mean age: 38.0 to 40.4 years). On day 4, the change from baseline in SARS-CoV-2 viral titer (log10 50% tissue culture infectious dose/mL) in patients with positive viral titer and viral RNA at baseline was greater with ensitrelvir 125 mg (mean [standard deviation], -2.42 [1.42]; P = 0.0712) and 250 mg (-2.81 [1.21]; P = 0.0083) versus placebo (-1.54 [0.74]); ensitrelvir treatment reduced SARS-CoV-2 RNA by -1.4 to -1.5 log10 copies/mL versus placebo. The viral titer and viral RNA were similar across groups on and after day 6. The median time to infectious viral clearance decreased by approximately 50 h with ensitrelvir treatment. All adverse events were mild to moderate. Ensitrelvir treatment demonstrated rapid SARS-CoV-2 clearance and was well tolerated (Japan Registry of Clinical Trials identifier: jRCT2031210350).

Effect of In Vitro Culture of Long Shoot Tip on Variant Structure and Titer of Grapevine Viruses.

Shoot tip culture is a very effective approach for studying plant viruses. In this study, we evaluated the numbers, diversity, and titer of grapevine viruses in in vitro grapevine plants after long shoot tip culture. Six virus-infected grapevine cultivars (Cabernet Franc, Cabernet Gernischt, Cabernet Sauvignon, Wink, Victoria, and Merlot) collected from six regions of China were used as the research materials. Approximately 1.5 cm long shoot tips were used for meristem culture. The average survival rate of the six grapevine cultivars was 45.7%. Merlot collected from Beijing showed the highest survival rate (80.0%). Regeneration was not achieved in Cabernet Gernischt collected from Liaoning province and Cabernet Sauvignon from Tianjin due to bacterial and fungal contamination. Virus detection conducted in the surviving regenerated plants showed that the virus infection status, including the viral numbers and the species present in plants grown in vitro, was the same as that in corresponding in vivo plants. Moreover, the analysis of sequence diversity and the mutation frequency in grapevine viruses in vitro indicated that the structure of grapevine viruses was stable in long shoot tip culture after four sub-culture passages. Further, the relative viral titer of in vitro grapevine plants was much higher than that of in vivo plants. These results aid in the investigation of viruses in woody plants.

Type and abundance of sialic acid receptors on host cell membrane affect infectivity and viral titer of different strains of Newcastle disease virus.

Cell-based vaccine manufacture is a very cost-effective approach for animal vaccine production. Newcastle disease virus (NDV) can infect a wide range of host cells, but the viral titers of cell culture are too low to meet the vaccine manufacture. In this study, we explored the selectivity of NDV vaccine strains LaSota and Mukteswar to type of sialic acid receptors and demonstrated the relationship between receptor expression levels on cell membrane and viral titers of cell culture. The results suggested that NDV strain LaSota preferentially binds to Neu5Ac-2-S-α-2,6 Gal10Me receptor (SAα2,6 Gal) and strain Mukteswar selectively binds to Neu5Ac-α-2,3 Gal-ß-1,4Glc (SAα2,3 Gal) receptor. Subsequently, the expression levels of SAα2,3 Gal and SAα2,6 Gal receptors on BHK-21 cell membrane were adjusted by overexpression and RNAi assays. The results indicated that the viral titers of NDV strains LaSota and Mukteswar in cell culture were positively correlated with the expression levels of SAα2,6 Gal and SAα2,3 Gal receptors on host cell membrane respectively. In conclusion, our studies provide an understanding of the relationship between infectivity of NDV different strains and receptor types of host cell, and provide a method to increase viral titer of NDV for cell-based vaccine production.

Exacerbation of Influenza A Virus Disease Severity by Respiratory Syncytial Virus Co-Infection in a Mouse Model.

Influenza A virus (IAV) and respiratory syncytial virus (RSV) are leading causes of childhood infections. RSV and influenza are competitive in vitro. In this study, the in vivo effects of RSV and IAV co-infection were investigated. Mice were intranasally inoculated with RSV, with IAV, or with both viruses (RSV+IAV and IAV+RSV) administered sequentially, 24 h apart. On days 3 and 7 post-infection, lung tissues were processed for viral loads and immune cell populations. Lung functions were also evaluated. Mortality was observed only in the IAV+RSV group (50% of mice did not survive beyond 7 days). On day 3, the viral loads in single-infected and co-infected mice were not significantly different. However, on day 7, the IAV titer was much higher in the IAV+RSV group, and the RSV viral load was reduced. CD4 T cells were reduced in all groups on day 7 except in single-infected mice. CD8 T cells were higher in all experimental groups except the RSV-alone group. Increased airway resistance and reduced thoracic compliance were demonstrated in both co-infected groups. This model indicates that, among all the infection types we studied, infection with IAV followed by RSV is associated with the highest IAV viral loads and the most morbidity and mortality.

ACoRE: Accurate SARS-CoV-2 genome reconstruction for the characterization of intra-host and inter-host viral diversity in clinical samples and for the evaluation of re-infections.

Sequencing the SARS-CoV-2 genome from clinical samples can be challenging, especially in specimens with low viral titer. Here we report Accurate SARS-CoV-2 genome Reconstruction (ACoRE), an amplicon-based viral genome sequencing workflow for the complete and accurate reconstruction of SARS-CoV-2 sequences from clinical samples, including suboptimal ones that would usually be excluded even if unique and irreplaceable. The protocol was optimized to improve flexibility and the combination of technical replicates was established as the central strategy to achieve accurate analysis of low-titer/suboptimal samples. We demonstrated the utility of the approach by achieving complete genome reconstruction and the identification of false-positive variants in >170 clinical samples, thus avoiding the generation of inaccurate and/or incomplete sequences. Most importantly, ACoRE was crucial to identify the correct viral strain responsible of a relapse case, that would be otherwise mis-classified as a re-infection due to missing or incorrect variant identification by a standard workflow.

High-throughput viral microneutralization method for feline coronavirus using image cytometry.

Feline coronaviruses (FCoV) are members of the alphacoronavirus genus that are further characterized by serotype (types I and II) based on the antigenicity of the spike (S) protein and by pathotype based on the associated clinical conditions. Feline enteric coronaviruses (FECV) are associated with the vast majority of infections and are typically asymptomatic. Within individual animals, FECV can mutate and cause a severe and usually fatal disease called feline infectious peritonitis (FIP), the leading infectious cause of death in domestic cat populations. There are no approved antiviral drugs or recommended vaccines to treat or prevent FCoV infection. The plaque reduction neutralization test (PRNT) traditionally employed to assess immune responses and to screen therapeutic and vaccine candidates is time-consuming, low-throughput, and typically requires 2-3 days for the formation and manual counting of cytolytic plaques. Host cells are capable of carrying heavy viral burden in the absence of visible cytolytic effects, thereby reducing the sensitivity of the assay. In addition, operator-to-operator variation can generate uncertainty in the results and digital records are not automatically created. To address these challenges we developed a novel high-throughput viral microneutralization assay, with quantification of virus-infected cells performed in a plate-based image cytometer. Host cell seeding density, microplate surface coating, virus concentration and incubation time, wash buffer and fluorescent labeling were optimized. Subsequently, this FCoV viral neutralization assay was used to explore immune correlates of protection using plasma from naturally FECV-infected cats. We demonstrate that the high-throughput viral neutralization assay using the Celigo Image Cytometer provides a robust and efficient method for the rapid screening of therapeutic antibodies, antiviral compounds, and vaccines. This method can be applied to various viral infectious diseases to accelerate vaccine and antiviral drug discovery and development.

EPA and DHA can modulate cell death via inhibition of the Fas/tBid-mediated signaling pathway with ISKNV infection in grouper fin cell line (GF-1) cells.

Polyunsaturated fatty acids (PUFAs) play important roles in organisms, including the structure and liquidity of cell membranes, anti-oxidation and anti-inflammation. Very little has been done in terms of the effect of PUFAs on cell death, especially on DNA virus. In this study, we demonstrated that the infectious spleen and kidney necrosis virus (ISKNV) can induce host cell death via the apoptotic cell death pathway, which correlated to modulation by PUFAs in grouper fin cell line (GF-1) cells. We screened the PUFAs, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), for the ability of different dosages to prevent cell death in GF-1 cells with ISKNV infection. In the results, each 10 µM of DHA and EPA treatment enhanced host cell viability up to 80% at day 5 post-infection. Then, in Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay, DHA- and EPA-treated groups reduced TUNEL positive signals 50% in GF-1 cells with ISKNV infection. Then, through studies of the mechanism of cell death, we found that ISKNV can induce both the Bax/caspase-3 and Fas/caspase-8/tBid death signaling pathways in GF-1 cells, especially at day 5 post-infection. Furthermore, we found that DHA and EPA treatment can either prevent caspase-3 activation on 17-kDa form cleavage or Bid cleaved (15-kDa form) for activation by caspase-8, apparently. On the other hand, the anti-apoptotic gene Bcl-2 was upregulated 0.3-fold and 0.15-fold at day 3 and day 5, respectively, compared to ISKNV-infected and DHA-treated cells; that this did not happen in the EPA-treated group showed that different PUFAs trigger different signals. Finally, ISKNV-infected GF-1 cells treated with either DHA or EPA showed a 5-fold difference in viral titer at day 5. Taken together, these results suggest that optimal PUFA treatment can affect cell death signaling through both the intrinsic and extrinsic death pathways, reducing viral expression and viral titer in GF-1 cells. This finding may provide insight in DNA virus infection and control.

High-Throughput Zika Viral Titer Assay for Rapid Screening of Antiviral Drugs.

Zika virus has recently emerged as a worldwide pathogen and public health burden due to its rapid spread and identification as a causative agent for multiple neurological defects, including congenital microcephaly. While there has been a flurry of recent research to address this emerging pathogen, there are currently no approved drug treatments for ZIKV infection. The gold standard for testing antiviral activity is to quantify infectious virion production. However, current infectious viral production assays, such as the plaque-forming or focus-forming unit assay, are tedious and labor intensive with a low-screening throughput. To facilitate drug development, we developed a Zika viral titration assay using an automated imaging system and an image analysis algorithm for viral colony quantification. This assay retained the principle of the classical virus titer assay, while improving workflow and offering higher screening throughput. In addition, this assay can be broadly adapted to quantification of other viruses.

Viral load and inflammatory cytokine dynamics associated with the prognosis of severe fever with thrombocytopenia syndrome virus infection: An autopsy case.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by a novel bunyavirus. The mechanism underlying disease progression remains unknown, and effective treatment strategy for SFTS is yet to be completely established, making its increasing incidence and subsequent mortality a great concern. Here, we present the autopsy case of a patient with rapidly progressed, fatal SFTS infection. Her viral titer and serum cytokines levels were measured daily and compared with the values of a survivor of the infection. Our findings elucidate the clinical features and pathophysiology of SFTS.

A proline insertion-deletion in the spike glycoprotein fusion peptide of mouse hepatitis virus strongly alters neuropathology.

Fusion peptides (FPs) in spike proteins are key players mediating early events in cell-to-cell fusion, vital for intercellular viral spread. A proline residue located at the central FP region has often been suggested to have a distinctive role in this fusion event. The spike glycoprotein from strain RSA59 (PP) of mouse hepatitis virus (MHV) contains two central, consecutive prolines in the FP. Here, we report that deletion of one of these proline residues, resulting in RSA59 (P), significantly affected neural cell syncytia formation and viral titers postinfection in vitro Transcranial inoculation of C57Bl/6 mice with RSA59 (PP) or RSA59 (P) yielded similar degrees of necrotizing hepatitis and meningitis, but only RSA59 (PP) produced widespread encephalitis that extended deeply into the brain parenchyma. By day 6 postinfection, both virus variants were mostly cleared from the brain. Interestingly, inoculation with the RSA59 (P)-carrying MHV significantly reduced demyelination at the chronic stage. We also found that the presence of two consecutive prolines in FP promotes a more ordered, compact, and rigid structure in the spike protein. These effects on FP structure were due to proline's unique stereochemical properties intrinsic to its secondary amino acid structure, revealed by molecular dynamics and NMR experiments. We therefore propose that the differences in the severity of encephalitis and demyelination between RSA59 (PP) and RSA59 (P) arise from the presence or absence, respectively, of the two consecutive prolines in FP. Our studies define a structural determinant of MHV entry in the brain parenchyma important for altered neuropathogenesis.

Seroprevalence, molecular epidemiology and quantitation of parvovirus B19 DNA levels in Iranian blood donors.

Human parvovirus B19 (B19) infection is common among blood donors, and healthy blood donors can transmit virus via transfusion. Due to resistance of B19 to viral inactivation methods, there is a potential concern regarding transfusion safety in blood products. We aimed to determine the seroprevalence, molecular epidemiology, and quantitation of B19 DNA levels in blood donors in Tehran, Iran. A total of 500 blood donors from Blood Transfusion Research Center were studied. ELISA was used for detection of B19 IgG and IgM and nested PCR was carried out for detection of B19 DNA. PCR products were subjected to direct sequencing. B19 viral load was determined by real time PCR. B19 IgG, IgM, and DNA were detected in 27.6, 2.6, and 1.2% of donors respectively. Ten samples (2%) were positive for both antibodies while in four cases (0.8%), B19 IgG and DNA detected simultaneously. One case had B19 IgM, IgG, and viremia concurrently. The titers of B19 DNA in four of six donors were more than 106 IU/mL (high level viremia) and all four cases had IgG simultaneously. All B19 isolates categorized in genotype 1A. Our findings indicated that prevalence of B19 DNA in Iranian blood donors was comparable with previous studies throughout the world. High level B19 viremia found in 0.8% of our donors and all viremic donors revealed neutralizing B19 antibody. Therefore implementation of a B19 screening test for each volunteer blood donor does not appear to be necessary but B19 testing for plasma-derived products seems important in Iranian donors.

Higher Cytopathic Effects of a Zika Virus Brazilian Isolate from Bahia Compared to a Canadian-Imported Thai Strain.

Zika virus (ZIKV) is an emerging pathogen from the Flaviviridae family. It represents a significant threat to global health due to its neurological and fetal pathogenesis (including microcephaly and congenital malformations), and its rapid dissemination across Latin America in recent years. The virus has spread from Africa to Asia, the Pacific islands and the Americas with limited knowledge about the pathogenesis associated with infection in recent years. Herein, we compared the ability of the Canadian-imported Thai strain PLCal_ZV and the Brazilian isolate HS-2015-BA-01 from Bahia to produce infectious ZIKV particles and cytopathic effects in a cell proliferation assay. We also compared the intracellular viral RNA accumulation of the two strains by quantitative RT-PCR (reverse transcription polymerase chain reaction) analyses. Our observations show that HS-2015-BA-01 is more cytopathic than PLCal_ZV in proliferation assays in Vero, Human Embryonic Kidney HEK 293T and neuroblastoma SH-SY5Y cells. Quantitative RT-PCR shows that the level of viral RNA is higher with HS-2015-BA-01 than with PLCal_ZV in two cell lines, but similar in a neuroblastoma cell line. The two strains have 13 amino acids polymorphisms and we analyzed their predicted protein secondary structure. The increased cytopathicity and RNA accumulation of the Brazilian ZIKV isolate compared to the Thai isolate could contribute to the increased pathogenicity observed during the Brazilian epidemic.

Mutations in HPV18 E1

The most highly expressed protein during the productive phase of the human papillomavirus (HPV) life cycle is E1^E4. Its full role during infection remains to be established. HPV E1^E4 is expressed during both the early and late stages of the virus life cycle and contributes to viral genome amplification. In an attempt to further outline the functions of E1^E4, and determine whether it plays a role in viral capsid assembly and viral infectivity, we examined wild-type E1^E4 as well as four E1^E4 truncation mutants. Our study revealed that HPV18 genomes containing the shortest truncated form of E1^E4, the 17/18 mutant, produced viral titers that were similar to wild-type virus and significantly higher compared to virions containing the three longer E1^E4 mutants. Additionally, the infectivity of virus containing the shortest E1^E4 mutation was equivalent to wild-type and significantly higher than the other three mutants. In contrast, infectivity was completely abrogated for virus containing the longer E1^E4 mutants, regardless of virion maturity. Taken together, our results indicate for the first time that HPV18 E1^E4 impacts capsid assembly and viral infectivity as well as virus maturation.

Relative abundance of deformed wing virus, Varroa destructor virus 1, and their recombinants in honey bees (Apis mellifera) assessed by kmer analysis of public RNA-Seq data.

Deformed wing virus (DWV) is a major pathogen of concern to apiculture, and recent reports have indicated the local predominance and potential virulence of recombinants between DWV and a related virus, Varroa destructor virus 1 (VDV). However, little is known about the frequency and titer of VDV and recombinants relative to DWV generally. In this study, I assessed the relative occurrence and titer of DWV and VDV in public RNA-seq accessions of honey bee using a rapid, kmer-based approach. Three recombinant types were detectable graphically and corroborated by de novo assembly. Recombination breakpoints did not disrupt the capsid-encoding region, consistent with previous reports, and both VDV- and DWV-derived capsids were observed in recombinant backgrounds. High abundance of VDV kmers was largely restricted to recombinant forms. Non-metric multidimensional scaling identified genotypic clusters among DWV isolates, which was corroborated by read mapping and consensus generation. The recently described DWV-C lineage was not detected in the searched accessions. The data further highlight the utility of high-throughput sequencing to monitor viral polymorphisms and statistically test biological predictors of titer, and point to the need for consistent methodologies and sampling schemes.

Identification of amino acid residues of the coat protein of Sri Lankan cassava mosaic virus affecting symptom production and viral titer in Nicotiana benthamiana.

Sri Lankan cassava mosaic virus (SLCMV) is bipartite begomovirus infecting cassava in India and Sri Lanka. Interestingly, the DNA-A component of the SLCMV alone is able to infect Nicotiana benthamiana causing symptoms of upward leaf rolling and stunting. One of the differences between monopartite and bipartite begomoviruses is the requirement of Coat Protein (CP) for infectivity; CP being essential for the former, but dispensable in the latter. This investigation was aimed to determine the importance of CP in the infectivity of the bipartite SLCMV, behaving as a monopartite virus in N. benthamiana. We tested CP-null mutants, single amino acid replacement mutants and double, triple and quadruple combinations of the above in SLCMV DNA-A, for infectivity, symptom development and viral DNA accumulation in N. benthamiana. While CP-null mutants were non-infectious, a majority of the single amino acid replacement mutants and their combinations retained infectivity, some with attenuated symptoms and reduced viral titers. Some of the combined mutations restored the attenuated symptoms to wild type levels. Some of the mutations were predicted to cause changes in the secondary structure of the CP, which roughly correlated with the attenuation of symptoms and the reduction in viral titers.