Virology

A SARS-CoV-2 lineage designated as P.3 with multiple signature mutations in the Spike protein region was recently reported with cases from the Central Visayas Region of the Philippines. Whole genome sequencing revealed that the 33 samples under this lineage all contain the E484K, N501Y, and P681H Spike mutations previously found in variants of concern (VOC) such as the B.1.351, the P.1 and B.1.1.7 variants first reported in South Africa, Brazil, and the United Kingdom, respectively. The possible implications of the mutations found in the Spike protein of P.3 were analyzed for their potential effects on structure, stability, and molecular surface character. The analysis suggests that these mutations could significantly impact the possible interactions of the Spike protein with the ACE2 receptor and neutralizing antibodies, and warrants further clinical investigation. Some of the mutations affecting the N and C terminal domains may have effects on Spike monomer and trimer stability. This report provides insights on relevant targets for the design of future diagnostics, therapeutics and vaccines against the evolving SARS-CoV-2 variants in the Philippines.

Infection with HIV is associated with an increased risk of cardiovascular disease (CVD), which may be mediated by the effect of the viral proteins, Nef and Tat, on inflammation and endothelial activation. The viral genes coding for Nef and Tat contain numerous polymorphisms, which we hypothesised may be differentially associated with endothelial activation. Therefore, our aim was to assess the association of these polymorphisms with endothelial activation and inflammation in subjects infected with HIV-1. The HIV-1 nef and tat genes were sequenced from clinical isolates from 31 and 34 patients, respectively. Plasma concentration of biomarkers of endothelial activation (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial leukocyte adhesion molecule-1 (E-selectin), monocyte chemoattractant protein-1 (MCP-1) and von Willebrand factor (vWF)), and biomarkers of inflammation (tumor necrosis factor- (TNF-), interleukin-6 (IL-6) and interleukin-8 (IL-8)), were measured. Analysis of HIV-1 nef gene sequences identified five polymorphisms (V16I, H40Y, T50A,H, S169N and H188Q,S) that were each significantly (p<0.05) associated with ICAM-1 plasma concentration. An additive effect of these variants on plasma ICAM-1 concentration (p=0.004 for trend), was observed. No significant associations were seen between Tat amino acid residues and plasma concentration of markers of endothelial activation and inflammation. These are the first human in vivo data that support the hypothesis that nef gene polymorphisms impact endothelial function. ImportanceCardiovascular disease (CVD) is a leading cause of mortality in adults living with HIV, which may in part be due to endothelial activation and inflammation caused by the viral proteins, Nef and Tat. However, there is no data from humans supporting the CVD-associated Nef and Tat hypothesis, and assays for accurately measuring Nef and Tat plasma concentrations are not currently available. Therefore, we hypothesized that polymorphisms in the nef and tat genes of clinical viral isolates may be associated with host plasma markers of endothelial activation and inflammation. Our results show that this was the case, with five nef polymorphisms showing both individual and additive association with plasma concentration of ICAM-1. The HIV-1 Tat gene, however, showed no significant association with plasma concentrations of markers of endothelial activation and inflammation. This is the first human study to directly link Nef to endothelial activation and to provide a possible screening tool i.e., nef genotyping, for identifying individuals at high risk of endothelial-based diseases.

Since their discovery, giant viruses have expanded our understanding of the principles of virology. Due to their gargantuan size and complexity, little is known about the life cycles of these viruses. To answer outstanding questions regarding giant virus infection mechanisms, we set out to determine biomolecular conditions that promote giant virus genome release. We generated four metastable infection intermediates in Samba virus (lineage A Mimiviridae) as visualized by cryo-EM, cryo-ET, and SEM. Each of these four intermediates reflects a stage that occurs in vivo. We show that these genome release stages are conserved in other, diverse giant viruses. Finally, we identified proteins that are released from Samba and newly discovered Tupanvirus through differential mass spectrometry. Our work revealed the molecular forces that trigger infection are conserved amongst disparate giant viruses. This study is also the first to identify specific proteins released during the initial stages of giant virus infection.

Antibodies are powerful tools to detect expressed proteins. However off-target recognition can confound their use. Therefore, careful characterization is needed to validate specificity in distinct applications. Here we report the sequence and characterization of a mouse recombinant antibody that specifically detects ORF46 of murine gammaherpesvirus 68 (MHV68). This ORF encodes the viral uracil DNA glycosylase (vUNG). The antibody does not recognize murine uracil DNA glycosylase and is useful in detecting vUNG expressed in virally infected cells. It can detect expressed vUNG in cells via immunostaining and microscopy or flow cytometry analysis. The antibody can detect vUNG from lysates of expressing cells via immunoblot under native conditions but not denaturing conditions. This suggests it recognizes a confirmational based epitope. Altogether this manuscript describes the utility of the anti-vUNG antibody and suitability for use in studies of MHV68 infected cells.

Gene therapy offers immense potential for treating various diseases, including cancer, immunodeficiencies, and cardiovascular conditions. The efficacy of gene therapy (GT) largely depends on the vector used for gene delivery. Viral vectors, while effective, pose risks including insertional mutagenesis, immune responses, and high manufacturing costs. Non-viral vectors, although safer and easier to produce, often exhibit lower transfection efficiency and weaker transgene expression. This highlights the need for novel, more efficient vectors. Among emerging strategies, bacteriophages are gaining attention as promising GT delivery vehicles due to their adaptability and safety profile. Filamentous phages like M13 have demonstrated potential as targeted gene delivery vectors. This study proposes constructing a single-stranded DNA (ssDNA) phage-based vector incorporating a eukaryotic gene cassette. By leveraging Ff phage replication mechanisms in E. coli, the study explores encapsidating ssDNA within anellovirus capsids. These small, ssDNA viruses, known for their ability to transfect diverse tissues, offer a safer alternative to conventional viral vectors. Through successful expression and assembly of anellovirus capsid proteins, ssDNA viral particles were produced ex vivo. This innovative E. coli-based anellovirus-phagemid system provides a promising, cost-effective platform for developing next-generation viral vectors in gene therapy.

The complete genome sequence of cotton virus A, a new virus infecting cotton (Gossypium spp.), was determined using high-throughput sequencing, PCR and rolling circle amplification. The 7,482-nt genome is a circular dsDNA molecule that codes for six open reading frames similar to other members of the Caulimovirus genus (family Caulimoviridae) but presenting key genome organization differences. P3, P5 and P6 are presumed movement, coat, and reverse transcriptase proteins, respectively. P1, P2 and P4 showed no homology to virus proteins. Endogenous viral elements were found in G. hirsutum and G. tomentosum, but not in G. barbadense, G. arboreum, or G. herbaceum.

Recent large-scale Ebola outbreaks, combined with improved follow-up of survivors, has permitted the observation of common long-term neurological sequelae in patients that have survived Ebola virus infection. To date there have been few studies into neurological infections by Ebola or related filoviruses, however, recent studies have isolated infectious virus from patients cerebrospinal fluid months after being discharged from the treatment facility. In order to determine whether different filoviruses were capable of infecting human neurons, the human neuroblastoma cell lines, SH-SY5Y and M17, were chemically-differentiated into more neuron-like cells using established protocols. The neuron-like profiles of the differentiated cells were confirmed by the determination of expression of a range of neuron-specific markers. Zaire ebolavirus, Reston ebolavirus, and Marburg virus were serially-passaged in both cell lines to determine permissiveness of the cells, as well as permit the acquisition of adaptive mutations in the viral genomes. Whilst Marburg virus grew to high titres in both cell lines, Zaire ebolavirus only grew in SH-SY5Y cells, and Reston ebolavirus rapidly died out in both cell lines. Whole-genome sequencing of the passaged viruses revealed two consensus-level non-coding mutations in the SH-SY5Y-passaged Marburg virus. Viral growth kinetics were determined for pre- and post-passaging Zaire ebolavirus and Marburg virus in both human neuronal cell lines, as well as the human hepatocyte cell line, Huh7. Growth kinetics were similar for both the pre- and post-passaged viruses, suggesting that adaptive mutations were not required for efficient growth in these cells. This study is the first to demonstrate that filoviruses are capable of infecting human neuron-like cells in a species-specific manner. Marburg virus-infected cells remained alive up to Day 21 post-infection, suggesting that long-term neurological sequelae following filovirus infection may be a result of direct neuronal infection, and that infection of neurons might contribute to viral persistence in survivors. Author SummaryFiloviruses, including Ebola and Marburg viruses, have been traditionally considered "haemorrhagic fever" viruses, with infections causing bleeding and frequently death. Recent large-scale outbreaks in Africa have challenged these assumptions due to a significant number of patients reporting neurological symptoms sometimes months after infection. In many of these patients, virus was present at detectable levels in the fluid surrounding the brain. There has been significant debate about the ability of Ebola and Marburg viruses to infect and grow in human neurons (brain cells), and evidence has been lacking due to the lack of feasibility in taking brain samples. Our study demonstrates that both Zaire ebolavirus and Marburg virus are capable of infecting cells derived from human brains without needing to change, and without killing the cells. Reston ebolavirus, a related virus that appears not to cause disease in humans, was not able to grow efficiently in these cells. Our findings show that these viruses might be capable of living in the brains of survivors for long periods of time, similar to previous observations in the eye and testes. In addition, the response of the body to these infected cells might account for the neurological symptoms described by patients.

The SARS-CoV-2 pandemic has reinforced efforts for developing effective vaccination strategy for existing and emerging viruses. Currently there are various vaccine technology available for treating viral diseases, however it is imperative to develop and investigate second-generation vaccines such as chimeric virus-like particles (chi-VLPs) vaccine for increased immunogenicity, ease of production and scalability to supplement the worldwide vaccine supply. Intriguingly, chi-VLPs expresses more than one antigenic epitope on its surface, hence it is expected to be a more effective vaccine candidate. Hereby, this study reports, a novel bivalent vaccine design of chimeric alphavirus coronavirus virus-like particles (ChAC-VLPs), displaying fusion glycoproteins of CHIKV and receptor binding domain (RBD) of SARS-CoV-2 on its surface. Uniqueness and versatility of ChAC-VLPs has been demonstrated via a various techniques including Western blot, Immunofluorescence, cryoEM, and dynamic light scattering (DLS). The multimeric epitope display of immunogenic antigens, i.e CHIKV envelop glycoprotein and SARS-CoV-2 RBD was validated by cell-based assays. ChAC-VLP immunized mice has shown substantial neutralization titres for CHIKV (PRNT50 of 1:25) from the serum collected after 2nd booster doses. Similarly, serum antibodies were detected for SARS-CoV2 RBD as observed by antigen specific ELISA and validated using surface plasmon resonance (SPR). SPR binding response was detected to be >200 RU for anti-RBD antibody in post-immunized mice sera. In conclusion, present study proposes ChAC-VLPs as a potential hybrid vaccine candidate for CHIKV and SARS-CoV-2 infection and contributes valuable insights in chi-VLPs domain.

ABSTRACTPlant viral infections pose a significant threat to global crop productivity. Despite their profound impact on agriculture, plant viruses have been relatively understudied, primarily due to technological limitations associated with classical molecular methods. However, the advent of NGS RNA-seq analysis has revolutionized virus characterization in environmental settings, overcoming previous limitations and providing a powerful tool for studying plant viruses. In an RNA-seq experiment conducted on a diseased Colombian Cannabis sativa hemp plant, we identified a linear single-stranded RNA (ssRNA) genome belonging to Tobacco Necrosis Virus A (TNVA), a common cause of necrotic lesions in plants such as tobacco and tulipa. The affected Cannabis sativa hemp plant exhibited severe symptoms, including alterations in pigmentation, leaf morphology such as chlorosis, necrotic tissue formation, and surface wear on the leaves. The complete genome sequence of the Cannabis sativa TNVA was 3,656 nucleotides long, containing five putative ORFs, and was classified in the family Tombusviridae, genus Alphanecrovirus, and belonging to the Necro-like clade based on RdRp protein phylogenetic analysis. Our analysis revealed a well-conserved RdRp protein among the Alphanecroviruses, with 89% of the amino acid residues in the peptide being entirely conserved. In contrast, the coat protein exhibited significantly higher variability, with only 49.3% of the residues being 100% conserved. Regarding the viral genome expression of Cannabis sativa TNVA, we observed that the virus was highly abundant in the leaves of the diseased plant, ranking among the topmost abundant transcripts, occupying the percentile position of 3.06%. Overall, our study generated the first reference genome of TNVA virus in the tropical region and reported the first case of this virus infecting a Cannabis sativa plant.

Despite being the predominant seal species in the Australian-New Zealand region and serving as a key indicator of marine environmental health, little is known about infectious diseases in New Zealand fur seals (Long-nosed fur seal; Arctocephalus forsteri). Several papillomaviruses have been identified in earless seals and sea lions, with the latter linked to cutaneous plaques and invasive squamous cell carcinoma. To date, no papillomaviruses have been reported in fur seals. We used traditional veterinary diagnostic techniques and metatranscriptomic sequencing of tissue samples to investigate the virome of New Zealand fur seals. We identified a novel papillomavirus, provisionally termed Arctocephalus forsteri papillomavirus 1 (AfPV1) in an animal with clinically and histologically identified oral papilloma-like lesions. RT-PCR confirmed the presence of AfPV1 only in oral papilloma samples from the affected individual. Phylogenetic analysis of the complete 7,926 bp genome of AfPV1 revealed that it clustered with taupapillomaviruses found in related Carnivora species. In addition, we identified the partial genome of a novel Gammaherpesvirus, Arctocephalus forsteri gammaherpesvirus 1 (AfGHV1), in a different individual without pathological evidence of viral infection. These findings highlight the need for further research into the disease associations and impact of undiagnosed and novel viruses on New Zealand fur seals.

The H7N9 influenza virus that emerged in 2013 is a dangerous infectious disease with a high mortality rate of up to 40%. Developing effective monoclonal antibodies (mAbs) to detect and treat the infection of this virus is therefore critical. In this study, we expressed hemagglutinin (HA) and neuraminidase (NA) of H7N9 (A/Anhui/1/2013) on the surface of baculovirus (i.e., HA7-Bac and NA9-Bac). Our results showed that both HA or NA proteins displayed on HA7-Bac or NA9-Bac could well maintain their native biological function. Mice antisera derived from the injections of either HA7-Bac- or NA9-Bac exhibited high inhibitory activity in the hemagglutination and neuraminidase assay of H7N9 virus. mAbs generated by immunization with HA7-Bac exhibited high neutralizing activity against H7N9 virus infectivity in cell assays, whereas mAbs generated by immunization with NA9-Bac inhibited neuraminidase activity. These results proved that baculovirus display of HA and NA from H7N9 could be convenient agents to generate neutralizing mAbs against virus infection.

Flaviviruses have emerged as major arthropod-transmitted pathogens and represent an increasing public health problem worldwide. High-throughput screening can be facilitated by the use of viruses that express easily detectable marker proteins. Developing molecular tools such as reporter-carrying versions of flaviviruses for studying viral replication and screening of antiviral compounds therefore represents a top priority. However, the engineering of flaviviruses carrying either fluorescent or luminescent reporters remains challenging due to the genetic instability caused by marker insertion; therefore, new approaches to overcome these limitations are needed. Here, we describe reverse genetic methods which includes design and validation of infectious clones of Zika, Kunjin and Dengue viruses harboring different reporter genes for infection, rescue, imaging and morphology using super-resolution microscopy. It was observed that for different flaviviruses constructs with identical design displayed strikingly different genetic stability while corresponding virions resembled wild-type virus particles in shape and size. A successful strategy was assessed to increase stability of rescued reporter virus and permit antiviral drug screening based on quantitative automated fluorescence microscopy and replication studies.

Shifts in land-use patterns and increased human-livestock-wildlife interactions have generated numerous possibilities for pathogen spillover. This demands increased efforts of pathogen surveillance in wildlife, especially in changing landscapes with high biodiversity. We investigated adenovirus diversity in small mammals, an understudied host taxon, from a forest-plantation mosaic in the Western Ghats biodiversity hotspot. Using PCR-based screening followed by Sanger sequencing and phylogenetic analyses, we attempted to detect and characterize adenovirus diversity in seven species of small mammals. We observed high prevalence (up to 38.8%) and identified five lineages of adenoviruses with unique mutations in the endemic and dominant small mammal species, Rattus satarae. These lineages significantly differed from other known Murine adenoviruses (p-distance > 25%), indicating the likelihood of novel adenovirus diversity in this endemic small mammal. Collectively, our results highlight the potential for unexplored diversity of DNA viruses like adenovirus in poorly explored host taxa inhabiting human-used landscapes and its zoonotic implications.

Ectoparasites play an important role in virus transmission among vertebrates. However, little is known about the extent and composition of viruses that pass between invertebrates and vertebrates. In Australia, flies and fleas support the mechanical transmission of viral biological controls against wild rabbits - rabbit haemorrhagic disease virus (RHDV) and myxoma virus. We compared virome structure and composition in rabbits and these associated ectoparasites, sequencing total RNA from multiple tissues and gut contents of wild rabbits, fleas collected from these rabbits, and flies trapped sympatrically. Meta-transcriptomic analyses identified 50 novel viruses from multiple RNA virus families. Rabbits and their ectoparasites were characterised by markedly different viromes: although viral contigs from six virus families/groups were found in both rabbits and ectoparasites, none were vertebrate-associated. A novel calicivirus and picornavirus detected in rabbit caecal content were vertebrate-specific: the newly detected calicivirus was distinct from known rabbit caliciviruses, while the novel picornavirus clustered with the Sapeloviruses. Several Picobirnaviridae were also identified, falling in diverse phylogenetic positions suggestive of an association with co-infecting bacteria. The remaining viruses found in rabbits, and all those from ectoparasites, were likely associated with invertebrates, plants and co-infecting endosymbionts. While no full genomes of vertebrate-associated viruses were detected in ectoparasites, suggestive of major barriers to biological transmission with active replication, small numbers of reads from rabbit astrovirus, RHDV and other lagoviruses were present in flies. This supports the role of flies in the mechanical transmission of RHDV and implies that they may assist the spread of astroviruses.

Chilli is an important vegetable and spice crop, is known to be infected by several viruses. Techniques used in diagnosis of plant viral diseases before Next-generation sequencing (NGS) are having limitation of identifying the only known viruses. In the present study, virome analyses in infected chilli leaf samples was carried out using NGS to know the diversity of both known and unknown viruses associated with diseased symptoms. For virome profiling, samples from 19 fields were collected from chilli plants showing leaf curling, vein banding, mosaic, mottling, shoestring/rat tail/filiform/leathery and dull coloured leaves. Viral disease incidence in the surveyed fields varied from 26.66% to 47.50%. Total RNA was extracted from the 19 chilli leaf samples collected from fields and were pooled at equimolar concentration for virome profiling. From the rRNA-depleted pooled total RNA, mRNA and sRNA libraries were prepared and sequenced using Illumina NOVASEQ 6000 platform. Raw sequence data obtained was de novo assembled using three approaches; mRNAome with Trinity, sRNAome with Velvet and whole transcriptome (WT) with SPAdes assembly. Chilli virome, pairwise sequence identity and phylogenetic analyses revealed the presence of seven different viruses; chilli leaf curl virus (ChiLCV) along with its associated alpha and betasatellites, cucumber mosaic virus (CMV), groundnut bud necrosis orthotospovirus (GBNV), pepper cryptic virus-2 (PCV-2), pepper vein yellows virus (PeVYV), bell pepper alphaendornavirus (BPEV) and tobacco vein clearing virus (TVCV). From the virus associated contigs, complete/near-complete genomes for ChiLCV, CMV, PCV-2, PeVYV and BPEV and, partial genomes for GBNV and TVCV were reconstructed from the RNAome. Recombination breakpoint analyses revealed presence of recombination breakpoints in ChiLCV (coat protein and AC4 regions), CMV RNA2 (2a protein region) and P0, P3 and P5 protein regions of PeVYV. viruses identified in the current study are known to be originated from intra and interspecific recombination. Further, all the viruses detected in the pooled RNA sample were validated by PCR and loop mediated isothermal amplification (LAMP) using specific primers designed. Among the seven viruses identified in the study in chilli, PeVYV and BPEV are the first reports from India.

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) is believed to have a zoonotic origin. Bats are a suspected natural host of SARS-CoV-2 because of sequence homology with other bat coronaviruses. Understanding the origin of the virus and determining species susceptibility is essential for managing the transmission potential during a pandemic. In a previous study, we established an in vitro animal model of SARS-CoV-2 susceptibility and replication in a non-permissive avian fibroblast cell line (DF1) based on expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) from different animal species. In this work, we express the ACE2 of seven bat species in DF1 cells and determine their ability to support attachment and replication of the original SARS-CoV-2 Wuhan lineage virus, as well as two variants, Delta and Lambda. We demonstrate that the ACE2 receptor of all seven species: little brown bat (Myotis lucifugus), great roundleaf bat (Hipposideros armiger), Pearsons horseshoe bat (Rhinolophus pearsonii), greater horseshoe bat (Rhinolophus ferrumequinum), Brazilian free-tailed bat (Tadarida brasiliensis), Egyptian rousette (Rousettus aegyptiacus), and Chinese rufous horseshoe bat (Rhinolophus sinicus), made the DF1 cells permissible to the three isolates of SARS-CoV-2. However, the level of virus replication differed between bat species and variant tested. In addition, the Wuhan lineage SARS-CoV-2 virus replicated to higher titers (104.5-105.5 TCID50) than either variant virus (103.5-104.5 TCID50) on pass 1. Interestingly, all viruses tested grew to higher titers (approximately 106 TCID50) when cells expressed the human ACE2 gene compared to bat ACE2. This study provides a practical in vitro method for further testing of animal species for potential susceptibility to current and emerging SARS-CoV-2 viruses.

The PCR amplification of majority of the ssRNA of both genomic and non-genomic mRNA is accomplished by RT-PCR. The mRNA is subjected to cDNA synthesis using reverse transcriptase and either Oligo(dT)18, or random or gene specific reverse primers based priming strategies. The choice of primer largely depends on the nature of 3 prime terminus of mRNA and length of cDNA synthesized. In general, oligo(dT)18 is the preferred choice for mRNAs having poly(A) tail at 3 prime terminus. In general, tobamoviruses lack any poly(A) tail at their 3 prime untranslated region (UTR) which forms a tRNA like structure and upstream pseudoknot domain except tow viruses viz., Hibiscus latent Fort Pierce virus (HLFPV) and Hibiscus latent Singapore virus (HLSV) which accommodate internal poly(A) sequences of 46 and 87 nucleotides long, respectively in their 3 prime UTR. However, determination of full nucleotide sequence of Pepper mild mottle virus (PMMoV) using an oligo(dT)18 primed cDNA as template indicated the libertinism of oligo(dT)18 in priming cDNA synthesis of RNA template which are known to lack poly(A) tail. at the end or internally at its 3 prime end. Moreover, coat protein (CP) gene of PMMoV and bean common mosaic virus (BCMV) (Potyvirus with a poly(A) tract at its 3 prime end) was amplified using cDNA primed with random primer as well as oligo(dT)18 was successfully amplified but with significant variation in the intensity of the amplification band in case of PMMoV but not in BCMV. This clearly indicated the presence of PMMoV mRNA with polyadenylated 3 prime tail in total RNA isolated from PMMoV infected capsicum leaves with abundance of non-polyadenylated PMMoV genomic RNA (gRNA). Hence, we hypothesize that the generation of polyadenylated RNA population during the infection cycle of PMMoV in pepper may be possible reason for libertinism of oligo(dT)18 in priming cDNA synthesis of RNA template isolated from PMMoV infected leaves followed by amplification of entire PMMoV genome through RT-PCR. This is first study indicating the presence of polyadenylated or polyadenylated rich regions in PMMoV gRNA acquired during the infection cycle in pepper.

We report the complete genome sequences of Penguin megriviruses from three different sources from Antarctica namely feces of Adelie penguin, feces of Weddell seal and ornithogenic soil. Phylogenetic analysis indicates the prevalence of very similar viruses in different sources of Antarctic environment. These genome sequences aid to understand the evolution of megriviruses in Antarctic ecology and reveal their place in global megrivirus phylogeny.

Vesicular stomatitis virus (VSV) is a promising oncolytic virus. Additionally, its glycoprotein G is the most commonly used envelope glycoprotein to pseudotype lentiviral vectors for gene therapy. However, G receptors (LDLR family members) are ubiquitous and expressed at the surface of non-target cells, precluding in vivo gene therapy. Recently, we identified G mutants that no longer bind to LDLR but retain their fusion activity. This opened the possibility of specifically retargeting the glycoprotein to receptors of interest. Here, we constructed chimeric glycoproteins fused with a nanobody at the amino-terminus of G. By experimental evolution, we identified two mutations in G improving the folding and functionality of chimeric Gs, regardless of the nanobody inserted at the amino-terminus. We then constructed chimeric glycoproteins using several nanobodies targeting HER2 receptor and, into these chimeras, we introduced mutations that abolish the recognition of LDL receptors. VSV and lentiviruses pseudotyped with these glycoproteins specifically infect cells expressing HER2. We have therefore identified G mutations that optimize the G scaffold to tolerate amino-terminal insertion of a nanobody and establish proof of concept that this approach can be used to confer a new tropism on G. This paves the way for targeted in vivo therapies.

Colorectal cancer (CRC) is a deadly disease with a high prevalence and mortality rate worldwide. Previous investigations have shown that Newcastle disease virus (NDV) exhibits oncolytic activity and antitumor immunostimulation properties on several types of tumor cells but not normal cells. This study aims to examine NDV oncolytic activity against two kinds of human CRC cell lines, i.e., HCT116 and SW620, as well as its ability to induce apoptosis. The results showed that CRC cell lines were susceptible to NDV LaSota strain and the mechanism of death was due to caspase-dependent pathways apoptosis, followed by interferon signaling competence. NDV-induced proinflammatory cytokines in CRC cells might have contributed to apoptosis mechanism. Therefore, further investigation is recommended, using the findings obtained in this study as a basis for an animal CRC model.