Passages In Vero Cells Research Articles

Mutations in the F protein of the live-attenuated respiratory syncytial virus vaccine candidate ΔNS2/Δ1313/I1314L increase the stability of infectivity and content of prefusion F protein.

Respiratory syncytial virus (RSV) is the leading viral cause of bronchiolitis and pneumonia in infants and toddlers, but there currently is no licensed pediatric vaccine. A leading vaccine candidate that has been evaluated for intranasal immunization in a recently completed phase 1/2 clinical trial is an attenuated version of RSV strain A2 called RSV/ΔNS2/Δ1313/I1314L (hereafter called ΔNS2). ΔNS2 is attenuated by deletion of the interferon antagonist NS2 gene and introduction into the L polymerase protein gene of a codon deletion (Δ1313) that confers temperature-sensitivity and is stabilized by a missense mutation (I1314L). Previously, introduction of four amino acid changes derived from a second RSV strain "line 19" (I79M, K191R, T357K, N371Y) into the F protein of strain A2 increased the stability of infectivity and the proportion of F protein in the highly immunogenic pre-fusion (pre-F) conformation. In the present study, these four "line 19" assignments were introduced into the ΔNS2 candidate, creating ΔNS2-L19F-4M. During in vitro growth in Vero cells, ΔNS2-L19F-4M had growth kinetics and peak titer similar to the ΔNS2 parent. ΔNS2-L19F-4M exhibited an enhanced proportion of pre-F protein, with a ratio of pre-F/total F that was 4.5- to 5.0-fold higher than that of the ΔNS2 parent. The stability of infectivity during incubation at 4°C, 25°C, 32°C and 37°C was greater for ΔNS2-L19F-4M; for example, after 28 days at 32°C, its titer was 100-fold greater than ΔNS2. ΔNS2-L19F-4M exhibited similar levels of replication in human airway epithelial (HAE) cells as ΔNS2. The four "line 19" F mutations were genetically stable during 10 rounds of serial passage in Vero cells. In African green monkeys, ΔNS2-L19F-4M and ΔNS2 had similar growth kinetics, peak titer, and immunogenicity. These results suggest that ΔNS2-L19F-4M is an improved live attenuated vaccine candidate whose enhanced stability may simplify its manufacture, storage and distribution, which merits further evaluation in a clinical trial in humans.

Detection and Genetic Analysis of Songling Virus in Haemaphysalis concinna near the China-North Korea Border

Background: Songling virus (SGLV) is a spherical, enveloped, fragmented, negative-stranded RNA virus belonging to the genus Orthonairovirus in the Nairoviridae family. SGLV is transmitted by ticks and can cause disease in humans. This study identified and characterized SGLV in Haemaphysalis concinna ticks collected in 2023 in the Yanbian Korean Autonomous Prefecture (China) near the China-North Korea border. Methods: A real-time quantitative polymerase chain reaction (RT-qPCR) was used to screen for SGLV nucleic acid in ticks. Baby hamster kidney (BHK-21) and African green monkey kidney (Vero) cells were used to isolate strains of SGLV from nucleic acid-positive samples through three successive passages. Next-generation sequencing and phylogenetics methods were used to characterize the SGLVs. Results: Of the 1659 ticks collected from 6 towns in the Yanbian Korean Autonomous Prefecture near the China-North Korea border, positive SGLV nucleic acid results were identified in 19 H. concinna tick pools from Helong and Longjing towns. This discovery led to the extraction of 17 SGLV genome sequences. Homology analysis that compare the newly discovered L, M, and S segments of SGLV strain HLJ1202 revealed nucleotide similarities ranging from 95.5%–97.1%, 91.9%–98.9%, and 98.3%–99.2%, respectively, and amino acid similarities ranging from 95.7%–97.4%, 97.1%–98.8%, and 98.2%–98.9%, respectively. Six distinct clades, characterized by specific geographic locations and host organisms, were identified on the Maximum Likelihood tree of the L segment. The YB129 and YB150 isolates demonstrated SGLV nucleic acid replication across three successive passages in Vero cells, as evidenced by the decrease in RT-qPCR Ct values. Conclusion: This study marks the initial identification of SGLV in H. concinna within the Yanbian Korean Autonomous Prefecture.

A novel live attenuated vaccine candidate protects chickens against subtype B avian metapneumovirus

Avian metapneumovirus (aMPV) is a highly contagious pathogen that causes acute upper respiratory tract diseases in chickens and turkeys, resulting in serious economic losses. Subtype B aMPV has recently become the dominant epidemic strain in China. We developed an attenuated aMPV subtype B strain by serial passaging in Vero cells and evaluated its safety and efficacy as a vaccine candidate. The safety test showed that after the 30th passage, the LN16-A strain was fully attenuated, as clinical signs of infection and histological lesions were absent after inoculation. The LN16-A strain did not revert to a virulent strain after five serial passages in chickens. The genomic sequence of LN16-A differed from that of the parent wild-type LN16 (wtLN16) strain and had nine amino acid mutations. In chickens, a single immunization with LN16-A induced robust humoral and cellular immune responses, including the abundant production of neutralizing antibodies, CD4+ T lymphocytes, and the Th1 (IFN-γ) and Th2 (IL-4 and IL-6) cytokines. We also confirmed that LN16-A provided 100% protection against subtype B aMPV and significantly reduced viral shedding and turbinate inflammation. Our findings suggest that the LN16-A strain is a promising live attenuated vaccine candidate that can prevent infection with subtype B aMPV.

Evaluation of the safety, immunogenicity and efficacy of a new live-attenuated lumpy skin disease vaccine in India

ABSTRACT Lumpy skin disease (LSD) was reported for the first time in India in 2019 and since then, it has become endemic. Since a homologous (LSD-virus based) vaccine was not available in the country, goatpox virus (GPV)-based heterologous vaccine was authorized for mass immunization to induce protection against LSD in cattle. This study describes the evaluation of safety, immunogenicity and efficacy of a new live-attenuated LSD vaccine developed by using an Indian field strain, isolated in 2019 from cattle. The virus was attenuated by continuous passage (P = 50) in Vero cells. The vaccine (50th LSDV passage in Vero cells, named as Lumpi-ProVac Ind ) did not induce any local or systemic reaction upon its experimental inoculation in calves (n = 10). At day 30 post-vaccination (pv), the vaccinated animals were shown to develop antibody- and cell-mediated immune responses and exhibited complete protection upon virulent LSDV challenge. A minimum Neethling response (0.018% animals; 5 out of 26,940 animals) of the vaccine was observed in the field trials conducted in 26,940 animals. There was no significant reduction in the milk yield in lactating animals (n = 10108), besides there was no abortion or any other reproductive disorder in the pregnant animals (n = 2889). Sero-conversion was observed in 85.18% animals in the field by day 30 pv.

Adaptation of the infectious bronchitis virus H120 vaccine strain to Vero cell lines

Infectious bronchitis virus (IBV) has restricted cell and tissue tropism. IBVs, except the Beaudette strain, can infect and replicate in chicken embryos, primary chicken embryo kidneys, and primary chicken kidney cells, only. The limited viral cell tropism of IBV substantially hinders in vitro cell-based research on pathogenic mechanisms and vaccine development. Herein, the parental H120 vaccine strain was serially passaged for five generations in chicken embryos, 20 passages in CK cells and 80 passages in Vero cells. This passaging yielded a Vero cell-adapted strain designated HV80. To further understand viral evolution, serial assessments of infection, replication, and transmission in Vero cells were performed for the viruses obtained every tenth passage. The ability to form syncytia and the replication efficiency significantly after the 50th passage (strain HV50). HV80 also displayed tropism extension to DF-1, BHK-21, HEK-293 T, and HeLa cells. Whole genome sequencing of viruses from every tenth generation revealed a total of 19 amino acid point mutations in the viral genome by passage 80, nine of which occurred in the S gene. The second furin cleavage site appeared in viral evolution and may be associated with cell tropism extension of HV80.

Development of cell culture based peste des petits ruminants (PPR) virus vaccine candidate from Bangladeshi isolates

This study was conducted to develop a cell culture based PPR virus vaccine candidate using recent Bangladeshi strain of peste des petits ruminant’s (PPR) virus. PPR virus was isolated from field outbreaks, confirmed by RT-PCR and used as viral inoculum for serial passaging in Vero cells for adaptation and attenuation. 60th serial passage had completed and RT-PCR and real time RT-PCR were done in every 5 passages for confirmation of PPR virus in tissue culture fluid (TCF). To assess the adaptation and attenuation cytopathology, virus titration, sequencing of both F and N genes and live animal experimentation were done. Different cellular alterations produced by PPR virus in infected Vero cells including syncytia formation, development of both intranuclear and intra cytoplasmic inclusion bodies and finally cell degradation are the indications of adaptation. The virus titre was found 2.5, 3.31, 3.55, 4.44, 4.71 and 6.5 Log10 TCID50/ml at 10th, 20th, 30th, 40th, 50th and 60th passages level respectively. In F gene sequence analysis it has been observed that few nucleotide (nt) and mino acid (aa) has been substituted as the effects of serial passaging of PPR virus in Vero cells. TCF at 60th passage level was found effective to produced protective antibody (Ab) titre in live animal experimentation. It is concluded that serially passaged and Vero cells adapted PPR virus TCF could be used as a vaccine candidate for further use to develop a potent & effective vaccine against PPR diseases.

Characterization and pathogenicity of the porcine epidemic diarrhea virus isolated in China.

Piglet diarrhea caused by the porcine epidemic diarrhea virus (PEDV) is a common problem on pig farms in China associated with high morbidity and mortality rates. In this study, three PEDV isolates were successfully detected after the fourth blind passage in Vero cells. The samples were obtained from infected piglet farms in Jilin (Changchun), and Shandong (Qingdao) Provinces of China and were designated as CH/CC-1/2018, CH/CC-2/2018, and CH/QD/2018. According to the analysis of the complete S protein gene sequence, the CH/CC-1/2018 and CH/CC-2/2018 were allocated to the G2b branch, while CH/QD/2018 was located in the G1a interval and was closer to the vaccine strain CV777. Successful detection and identification of the isolated strains were carried out using electron microscopy and indirect immunofluorescence. Meanwhile, animal challenge experiments and viral RNA copies determination were used to compare the pathogenicity. The results showed that CH/CC-1/2018 in Changchun was more pathogenic than CH/QD/2018 in Qingdao. In conclusion, the discovery of these new strains is conducive to the development of vaccines to prevent the pandemic of PEDV, especially that the CH/CC-1/2018, and CH/CC-2/2018 were not related to the classical vaccine strain CV777.

Adaptation of the sheep pox virus (Poxviridae: <i>Capripoxvirus: Sheeppox virus</i>) to African green monkey kidney cell line and evaluation of its immunobiological properties

Outbreaks of infectious diseases seriously hinder the preservation and increase of the number of small ruminants. Such infections include sheep pox virus (SPPV). According to the OIE data of 2021, SPP outbreaks were registered in countries such as Turkey, Israel, China, Maldives, Mongolia, Thailand, Russia, Algeria, Kenya, and in 2019 in Mangistau and Atyrau regions. In Kazakhstan annually conducts routine immunization of sheep at risk with a live attenuated vaccine produced by RIBSP. The object of the study was the vaccine strain of NISHI and the virulent strain A of the sheep pox virus. The virus was propagated in Vero cells. To determine the harmlessness and immunogenicity, sheep of the Kazakh fine-wool breed aged from 6 to 12 months were used. Virological, serological and immunobiological methods were used in the study. The results of the adaptation of the NISHI strain of SPPV to the Vero cell line are presented. Five passages in Vero cells resulted to the adaptation of the NISHI strain with the manifestation of a cytopathogenic effect specific to SPPV with a titer of 6.50 lg TCD50/ml. Following immunization, the formation of immunity was observed in animals on day 7 with an average protective titer 1.8 log2, which increased by day 21 to 4.33 log2. It has been established that the NISHI strain of SPPV retains its virological and immunobiological properties during reproduction in a Vero cell line.

Characterization of avain metapneumovirus subgroup C isolated from chickens in Beijing, China

Avian metapneumovirus (aMPV) is an important causative agent that causes acute respiratory disease and egg-dropping in chickens and turkeys. Here, we characterized an aMPV subgroup C (aMPV/C) from 320-day-old broiler breeder chickens with severe respiratory diseases in Beijing, China, as evidenced by RT-PCR typing and confirmation of the nucleoprotein (N) gene sequence. The N gene sequence of the aMPV/C strain (designated BJ17) exhibited no deletions or insertions and possessed 94.6% to 99.6% identity to those of published aMPV/C isolates. The phylogenetic tree of the nucleotide sequences constructed using the neighbor-joining clustering method showed that the BJ17 strain formed one cluster with other aMPV/C viruses and formed one subcluster with published Chinese aMPV/C isolates regardless of Muscovy duck or chicken origins. Comparative analysis of the N proteins showed that a unique amino acid residue D at position 110 might be associated with regional distribution due to its occurrence in all the Chinese aMPV/C isolates only. Strain BJ17 was successfully isolated by cultured Vero cell passage and further inoculated in 3-wk-old specific-pathogen-free chickens for the examination of pathogenicity. Animal experimental results showed that BJ17-inoculated chickens had severe respiratory diseases and inflammatory lesions, as demonstrated by pathological changes and aMPV antigen in the nasal turbinate, tracheae, and lung tissues. These results enrich the available information regarding the epidemiology and pathogenicity of aMPV/C in chickens, which may facilitate the development of effective measures against aMPV/C infection in China.

Rhinolophus sinicus virome revealed multiple novel mosquito-borne zoonotic viruses.

To exploit the Rhinolophus sinicus–specific virome, 29 Rhinolophus sinicus were gathered in Lincang, China. Enriched viral sequences of 22 virus families were acquired by metavirome techniques. Hereby, the part of virome in Rhinolophus sinicus, including Chikungunya virus (CHIKV), Getah virus, and Japanese encephalitis virus (JEV) were validated by PCR. Five CHIKV viral sequences were amplified, among which CHIKV-China/B2016C-1 shared the highest homology to CHIKV isolated from Italy in 2007, with the genotype as African ECS. Eight JEV viral sequences were amplified, of which JEV-China/B2016E-1 shared the highest homology with at least 91.3% nt identity with the JEV sequence found in South Korea in 1988 and was classified as genotype III. Notably, JEV was isolated for the first time in Rhinolophus sinicus. The newly isolated JEV-China/B2016-1 could increase infectivity while passaging in Vero cells from BHK-21 cells. Overall, the research sheds insight into the diversity and viral susceptibility dynamics of the virome in Rhinolophus sinicus and reveals new light on the ecology of other important viral hosts.

Attenuation and genetic characteristics of a Moroccan strain of Camel pox virus

Camel pox (CML) is a widespread infectious viral disease of camels that causes huge economic losses to the camel industry. In this study, a local strain of Camel pox virus (CMLV) was attenuated by 175 serial passages in Vero cells and the residual pathogenicity and infectivity were tested in naïve camels at 120, 150 and 175 passage levels. Also, the safety and immunogenicity of the 175th passage were evaluated in camels using a dose of 104.0 Tissue Culture Dose 50% (TCID50) and monitored for up to one-year post vaccination (pv) for neutralizing antibody. Seroconversion was noted at day 14 pv with neutralizing antibody titers ranging from 0.5 and 1.6 logs over the one-year of the study. Among 8 camels inoculated with the P175 strain, 4 were challenged at 12-month pv with 105.7 TCID50/ml of the original virulent CMLV and complete protection was recorded in all animals. Whole genome sequencing detected six mutations in the original CMLV strain that were not present in the attenuated 175th passage of this strain. Overall, the findings of this study indicated that the 175th passage of the CMLV was attenuated, safe and afforded protection to camels against virulent CMLV, and is therefore, a promising vaccine candidate for the prevention of CML in camels.

Metavirome Analysis of Culex tritaeniorhynchus Reveals Novel Japanese Encephalitis Virus and Chikungunya Virus.

To explore the Culex tritaeniorhynchuses–specific virome, 6400 C. tritaeniorhynchuses were collected in Honghe autonomous prefecture, China. Abundant virus sequences were obtained from 28 viral families using metavirome sequencing. Herein, several viruses in C. tritaeniorhynchuses virome were verified using the PCR technique, which covers Japanese encephalitis virus (JEV), Getah virus, and even Chikungunya virus (CHIKV). Seven JEV gene sequences were amplified successfully, of which JEV-China/CT2016E-1 shared the highest homology with the known JEV sequence isolated in Korea, 1946, with at least 96.1% nucleotide (nt) identity, which belonged to genotype III. Nine CHIKV gene sequences were amplified, which shared the highest with at least 93.0% nt identity with CHIKV from Thailand isolated in 2007, which was assigned to genotype Asian. Remarkably, CHIKV was isolated from C. tritaeniorhynchus in China for the first time. It was initially confirmed that the isolated virus CHIKV-China/CT2016-1 may increase infectivity after passaging in Vero cells from BHK-21 cells. Collectively, our study reveals the diversity, properties, and potential virus susceptibility dynamics of the C. tritaeniorhynchus virome and sheds new perspectives on the viral ecology in other important biological vectors.

Effective cross-protection of a lyophilized live gE/gI/TK-deleted pseudorabies virus (PRV) vaccine against classical and variant PRV challenges

Classical Bartha-K61 strains could not provide complete protection against the emerging highly virulent pseudorabies virus (PRV) variant strains, which has caused great economic losses to swine industry in China. In this study, a gE/gI/TK-deleted PRV vaccine strain based on a circulating PRV variant strain HeB12 was generated by serial passages in Vero cells and a lyophilized formulation was prepared as a live-attenuated PRV vaccine. Compared to commercial Bartha-K61 strains, vaccine efficacy in vivo experiments showed that the novel triple gene-deleted variant vaccine could provide complete cross-protection against the lethal challenges by the classical RA strain and variant TJ12 strain, indicating that it could be a better alternative product than the currently used Bartha-K61 strains for the control and eradication of epidemic pseudorabies in China.

High-Titer Self-Propagating Capsidless Chikungunya Virus Generated in Vero Cells as a Strategy for Alphavirus Vaccine Development.

In our previous study, we found that a new type of Chikungunya virus particle with a complete capsid deletion (ΔC-CHIKV) is still infectious in BHK-21 cells and demonstrated its potential as a live attenuated vaccine candidate. However, the low yield as well as the disability to propagate in vaccine production cell line Vero of ΔC-CHIKV are not practical for commercial vaccine development. In this study, we not only achieved the successful propagation of the viral particle in Vero cells, but significantly improved its yield through construction of a chimeric VEEV-ΔC-CHIKV and extensive passage in Vero cells. Mechanistically, high production of VEEV-ΔC-CHIKV is due to the improvement of viral RNA packaging efficiency conferred by adaptive mutations, especially those in envelope proteins. Similar to ΔC-CHIKV, the passaged VEEV-ΔC-CHIKV is safe, immunogenic, and efficacious, which protects mice from CHIKV challenge after only one shot of immunization. Our study demonstrates that the utilization of infectious capsidless viral particle of CHIKV as a vaccine candidate is a practical strategy for the development of alphavirus vaccine. IMPORTANCE Chikungunya virus (CHIKV) is one of important emerging alphaviruses. Currently, there are no licensed vaccines against CHIKV infection. We have previously found a new type of Chikungunya virus particle with a complete capsid deletion (ΔC-CHIKV) as a live attenuated vaccine candidate that is not suitable for commercial vaccine development with the low viral titer production. In this study, we significantly improved its production through construction of a chimeric VEEV-ΔC-CHIKV. Our results proved the utilization of infectious capsidless viral particle of CHIKV as a safe and practical vaccine candidate.

Mutation rate of SARS-CoV-2 and emergence of mutators during experimental evolution.

Background and objectivesTo understand how organisms evolve, it is fundamental to study how mutations emerge and establish. Here, we estimated the rate of mutation accumulation of SARS-CoV-2 in vitro and investigated the repeatability of its evolution when facing a new cell type but no immune or drug pressures.MethodologyWe performed experimental evolution with two strains of SARS-CoV-2, one carrying the originally described spike protein (CoV-2-D) and another carrying the D614G mutation that has spread worldwide (CoV-2-G). After 15 passages in Vero cells and whole genome sequencing, we characterized the spectrum and rate of the emerging mutations and looked for evidences of selection across the genomes of both strains.ResultsFrom the frequencies of the mutations accumulated, and excluding the genes with signals of selection, we estimate a spontaneous mutation rate of 1.3 × 10−6 ± 0.2 × 10−6 per-base per-infection cycle (mean across both lineages of SARS-CoV-2 ± 2SEM). We further show that mutation accumulation is larger in the CoV-2-D lineage and heterogeneous along the genome, consistent with the action of positive selection on the spike protein, which accumulated five times more mutations than the corresponding genomic average. We also observe the emergence of mutators in the CoV-2-G background, likely linked to mutations in the RNA-dependent RNA polymerase and/or in the error-correcting exonuclease protein.Conclusions and implicationsThese results provide valuable information on how spontaneous mutations emerge in SARS-CoV-2 and on how selection can shape its genome toward adaptation to new environments. Lay Summary: Each time a virus replicates inside a cell, errors (mutations) occur. Here, via laboratory propagation in cells originally isolated from the kidney epithelium of African green monkeys, we estimated the rate at which the SARS-CoV-2 virus mutates—an important parameter for understanding how it can evolve within and across humans. We also confirm the potential of its Spike protein to adapt to a new environment and report the emergence of mutators—viral populations where mutations occur at a significantly faster rate.

Abstract P-44: SARS-CoV-2 Inactivation by Ultraviolet Light Does Not Violate Virus Morphology, Antigenic and Immunogenic Properties

Background: Vaccination is the most effective tool for reducing the morbidity and mortality of COVID-19. Particular interest is the development of whole-virion inactivated vaccines, since such vaccines include a complete set of viral structural proteins. One of the requirements for the whole-virion vaccines is the guarantee of complete virus inactivation while maintaining the native conformation of protective antigens. The aim of this study is to evaluate the effect of ultraviolet (UV) treatment on the morphology, antigenic and immunogenic properties of the SARS-CoV-2. Methods: SARS-CoV-2, strain "Dubrovka" (GenBank No.: MW514307.1), grown in Vero CCL81 cell culture (ATCC). Viral reproduction was monitored by real-time qRT-PCR, ELISA and virus titration in Vero cells. Virus was inactivated by treating with ultraviolet light for 5 minutes using a standard biosafety cabinet UV irradiator. Virus inactivation control was performed by three "blind" passages in Vero cells. Clarified viral material was concentrated on Amicon MWCO 100 kDa (Millipore) columns. Negatively stained with uranyl acetate viral preparation was examined by transmission electron microscopy (TEM). Mice were immunized with a UV-inactivated virus subcutaneously in two variants (5 animals per group) - with and without Freund's adjuvant, twice with an interval between immunizations of 2 weeks. The titer of virus-neutralizing antibodies in mouse sera was determined in Vero cell culture. Results: Preparation of the SARS-CoV-2 coronavirus with a titer of 7.5 lg TCID50/ml and concentration of viral RNA of 8.5 lg copies/ml was obtained in Vero cells. After UV-treatment the presence in the prepapation of SARS-CoV-2 antigen was confirmed by ELISA with a set of COVID-19 convalescent sera. The particles with coronavirus morphodiagnostic signs were imaged by TEM - rounded shape with characteristic spikes of 12-15 nm on the envelope, the diameter of the virion was 90-110 nm. Neutralizing antibodies were detected in the sera of all immunized mice, whereas in animals of the control group neutralizing antibodies were not detected. Neutralizing antibodies titer was significantly higher in animals immunized by a virus with Freund's adjuvant - on average 448, than without adjuvant - 64 (p<0.01). Conclusion: Treatment of SARS-CoV-2 by UV light completely inactivates its infectivity, while retaining the typical coronavirus virions morphology, antigenic properties, and ability to induce in mice a synthesis of neutralizing antibodies.

Live attenuated viral vectors based on the rubella vaccine strain RA27/3

Abstract We have developed a series of live viral vectors based on the live attenuated rubella vaccine strain RA27/3. These vectors can express a variety of heterologous vaccine antigens without interfering with vector growth or stable antigen expression. The vaccine inserts were expressed for at least 5 to 10 passages in Vero cells. Proteins as large as the ectodomain of HIV gp140 could be expressed (90 kDa), and they retained the native conformation. Recently expressed vaccine inserts include: P27 Gag protein of SIV, the NANP repeat sequence of malaria circumsporozoite protein, and the receptor binding domain of Sars CoV-2 S1 spike protein. We have studied these vectors for immunogenicity in a rhesus macaque model. The rubella vectors are based on the rubella vaccine strain, for which one or two doses can elicit lifelong immunity. The vectors are a little different: The best immune response was observed when the macaques were primed with a DNA vaccine, followed by rubella vectors, or when they were primed with rubella vector and then boosted with the native protein. In both cases, an immunization strategy with only one exposure to rubella antigens was better than giving it twice. This could occur if the secondary response to rubella antigens inhibited the response to the vaccine insert. Alternatively, the strong anamnestic response to a boost of protein antigens suggests that the level of protein expression by the vector was the limiting factor. In future studies, we will test whether vectors with better growth characteristics and stronger antigen expression levels can improve antibody titers, elicit neutralizing antibodies, and protect against a viral challenge.

RNA Sequencing Data Sets and Their Whole-Genome Sequence Assembly of Dengue Virus from Three Serial Passages in Vero Cells.

We present RNA sequencing data sets and their genome sequence assembly for dengue virus that was isolated from a patient with dengue hemorrhagic fever and serially propagated in Vero cells. RNA sequencing data obtained from the first, third, and fifth passages and their corresponding whole-genome sequences are provided in this work.

Studies on co-infection of Peste des petits ruminants virus and orf virus In vitro

In peste des petits ruminants (PPR) affected goat/sheep flocks the most commonly occurring mixed viral infection is orf. Hence the present study was aimed to study the interaction of PPR virus (PPRV) and orf virus by co-infecting both the viruses in vitro using Vero cells. Both the PPRV field isolate and orf virus field isolate, adapted in Vero cells were utilized for co-infection. Equal quantum of both the viruses (100 CCID50) was used for all the three methods of co-infection (Initial infection with PPRV virus followed by orf virus, Initial infection with orf virus followed by PPR Virus and Simultaneous infection with both the PPR virus and orf virus). Three types of control i.e. PPRV infected Vero cells, orf infected Vero cells and uninfected Vero cells were maintained. In all the three types of co-infection of PPRV and orf virus, the results revealed only the establishment of PPRV infection in Vero cells which was detected even at 5th passage whereas, the co-infected orf virus was not detected after second passage. Both the orf virus and PPRV were detected in all the five passages of their virus control Vero cells infected separately with respective viruses. The PPRV has dominated the co-infected orf virus and established the infection even at 5th passage in Vero cells.

Long-term persistence of infectious Zika virus: Inflammation and behavioral sequela in mice.

The neurodevelopmental defects associated with ZIKV infections early in pregnancy are well documented, however the potential defects and long-term consequences associated with milder infections in late pregnancy and perinatal period are less well understood. To model these, we challenged 1 day old (P1) immunocompetent C57BL/6 mice with ZIKV. The animals developed a transient neurological syndrome including unsteady gait, kinetic tremors, severe ataxia and seizures 10–15 days post-infection (dpi) but symptoms subsided after a week, and most animals survived. Despite apparent recovery, MRI of convalescent mice show reduced cerebellar volume that correlates with altered coordination and motor function as well as hyperactivity and impulsivity. Persistent mRNA levels of pro-inflammatory genes including Cd80, Il-1α, and Ifn-γ together with Cd3, Cd8 and perforin (PrfA), suggested persistence of low-grade inflammation. Surprisingly, the brain parenchyma of convalescent mice harbor multiple small discrete foci with viral antigen, active apoptotic processes in neurons, and cellular infiltrates, surrounded by activated astrocytes and microglia as late as 1-year post-infection. Detection of negative-sense strand viral RNA and isolation of infectious virus derived from these convalescent mice by blinded passage in Vero cells confirmed long-term persistence of replicating ZIKV in CNS of convalescent mice. Although the infection appears to persist in defined reservoirs within CNS, the resulting inflammation could increase the risk of neurodegenerative disorders. This raises concern regarding possible long-term effects in asymptomatic children exposed to the virus and suggests that long-term neurological and behavioral monitoring as well as anti-viral treatment to clear virus from the CNS may be useful in patients exposed to ZIKV at an early age.