High Viral Titers Research Articles

Study on the infection characteristics and pathogenesis of duck circovirus 1 in Cherry Valley meat ducks.

Duck circovirus (DuCV) infected multiple breeds of ducks and was widespread in duck factories worldwide. Infected ducks exhibited feathering disorder, growth retardation and immunosuppression, which lead to secondary infection with other pathogens easily. But till now, there has been little research on the study of DuCV due to the absence of appropriate cultural measures. This study developed an animal model to investigate the pathogenesis of DuCV-1 in vivo by infecting 84 one-day-old Cherry Valley meat ducklings. At 1, 3, 7, 14, 21, 28 and 35 days post-infection (DPI), six Cherry Valley meat ducks were randomly chosen to detect some indexes. The results showed that ducks infected with DuCV-1 exhibited depression, feather disorders and gained weight slowly. DuCV-1 was detected in serum, cloacal swabs, and throat swabs at 1 DPI and sustained during the experimental period. The contents of calcium, phosphorus and lymphocytes decreased while alanine aminotransferase and total bilirubin increased in the serum of sick ducks. DuCV-1 was detectable in all organs and caused multiple local lesions. The virus was initially detected in the liver and the organ with the highest viral titer was the thymus. Besides, immune organ indexes were significantly decreased. These results demonstrated that DuCV-1 could be transmitted horizontally and cause infection persistently. It possesses broad tissue tropism, damaging immune organs and potentially inducing immune suppression. This study could provide references for future research on the pathogenic mechanism of DuCV.

Pasteurisation temperatures effectively inactivate influenza A viruses in milk

In late 2023 an H5N1 lineage of high pathogenicity avian influenza virus (HPAIV) began circulating in American dairy cattle Concerningly, high titres of virus were detected in cows’ milk, raising the concern that milk could be a route of human infection. Cows’ milk is typically pasteurised to render it safe for human consumption, but the effectiveness of pasteurisation on influenza viruses in milk was uncertain. To assess this, here we evaluate heat inactivation in milk for a panel of different influenza viruses. This includes human and avian influenza A viruses (IAVs), an influenza D virus that naturally infects cattle, and recombinant IAVs carrying contemporary avian or bovine H5N1 glycoproteins. At pasteurisation temperatures of 63 °C and 72 °C, we find that viral infectivity is rapidly lost and becomes undetectable before the times recommended for pasteurisation (30 minutes and 15 seconds, respectively). We then show that an H5N1 HPAIV in milk is effectively inactivated by a comparable treatment, even though its genetic material remains detectable. We conclude that pasteurisation conditions should effectively inactivate H5N1 HPAIV in cows’ milk, but that unpasteurised milk could carry infectious influenza viruses.

Resistance-breaking strains of tomato spotted wilt virus hamper photosynthesis and protein synthesis pathways in a virus accumulation-dependent manner in Sw5-carrying tomatoes

Tomato spotted wilt virus (TSWV; Orthotospovirus tomatomaculae) is one of the major horticultural threats due to its worldwide distribution and broad host range. In Italy, TSWV is widely spread in tomato (Solanum lycopersicum) crops and causes severe yield losses. In the last decades, several tomato varieties carrying the Sw-5b gene for resistance to TSWV have been released. We investigated the interaction between Sw-5b-carrying tomatoes and Sw5-Resistance-Breaking (SRB) TSWV to elucidate the molecular mechanisms underlying resistance breakage. Transcriptome sequencing (RNA-Seq) was used to analyze 18 tomato leaf samples collected from a field crop naturally infected by SRB TSWV in Italy. An increase in virus accumulation level in leaf tissues (titer) resulted in a higher number of differentially expressed genes (DEGs), ranging from 33 to 44% of the whole transcriptome, when the samples with the lowest and the highest virus titer were compared to the asymptomatic sample, respectively. Photosynthesis and protein biosynthesis were the main down-regulated biological processes, while enzyme families such as oxidoreductases and transferases, genes related to the response to biotic stimuli, solute transport, and vesicle trafficking were overall up-regulated. Remarkably, the expression of around 45% of genes (ca. 14000) of the whole transcriptome was significantly (P < 0.05) correlated (positively or negatively) to the virus titer, and in 6% of cases (about 2000 genes) the correlation was high (i.e., absolute value of R2 > 0.85). This phenomenon was also verified on 15 genes by a quantitative reverse transcription PCR assay on a greenhouse experiment with a different Sw-5b-tomato variety artificially inoculated with another SRB TSWV strain. In conclusion, the tomato transcriptome was considerably rearranged upon TSWV infection, with deregulation of photosynthesis, protein biosynthesis, and induction of defense pathways. Finally, this research demonstrated that the magnitude of transcriptional changes was proportional to the virus accumulation level in the leaves.

Protocol for recombinant rabies virus titration by quantitative PCR.

Preparing high-titer virus and performing accurate titer determination are criticalto subsequent experiments. However, not all applied recombinant rabies viruses, such as the L-deleted virus,1 are equipped with fluorescent proteins for titration by fluorescence-activated cell sorting (FACS). Here, we present a quantitative reverse-transcription PCR (RT-qPCR) approach for titrating recombinant rabies virus. We describe steps for preparing standards for RT-qPCR, rabies virus genome RNA extraction, and reverse transcription of virus RNA. We then detail procedures for RT-qPCR for titration and stereotaxic rabies virus injection for titer verification.

Pathogenesis of bovine H5N1 clade 2.3.4.4b infection in Macaques.

Since early 2022 highly pathogenic avian influenza (HPAI) H5N1 virus infections have been reported in wild aquatic birds and poultry throughout the United States (US) with spillover into several mammalian species1-6. In March 2024, HPAIV H5N1 clade 2.3.4.4b was first detected in dairy cows in Texas, US and continues to circulate on dairy farms in multiple states7,8. Milk production and quality are diminished in infected dairy cows, with high virus titers in milk raising concerns of exposure to mammals including humans through consumption9-12. Here we investigated routes of infection with bovine HPAIV H5N1 clade 2.3.4.4b in cynomolgus macaques, a surrogate model for human infection13. We show that intranasal or intratracheal inoculation of macaques could cause systemic infection resulting in mild and severe respiratory disease, respectively. In contrast, infection by the orogastric route resulted in limited infection and seroconversion of macaques which remained subclinical.

Enhanced Porcine Reproductive and Respiratory Syndrome Virus Replication in Nsp4- or Nsp2-Overexpressed Marc-145 Cell Lines.

Porcine reproductive and respiratory syndrome (PRRS) causes significant economic losses to the swine industry. The killed PRRSV vaccine has been reported to be safe and could elicit humoral responses. The killed PRRSV vaccine with a high viral antigen load combined with robust adjuvants could provide good protection against the infection. However, the high virus titer of PRRSV on the successive production cell lines is the prerequisite for this strategy. In this study, we explored PRRSV production in two recombinant Marc-145 cell lines expressing Nsp2 or Nsp4 through a lentivirus system. The results demonstrated that either Nsp2 or Nsp4 expressing Marc-145 cells did not affect cell morphology and growth kinetics but significantly enhanced PRRSV replication. Overall, our exploration may enable the production of high-yield PRRSV and offer a potential tool for developing safer and more effective PRRSV vaccines.

Epithelial-to-mesenchymal transition and live cell extrusion contribute to measles virus release from human airway epithelia.

Measles virus (MeV) is a highly contagious respiratory virus transmitted via aerosols. To understand how MeV exits the airways of an infected host, we use unpassaged primary cultures of human airway epithelial cells (HAE). MeV typically remains cell-associated in HAE and forms foci of infection, termed infectious centers, by directly spreading cell-to-cell. We previously described the phenomenon in which infectious centers detach en masse from HAE and remain viable. Here, we investigate the mechanism of this cellular detachment. Via immunostaining, we observed loss of tight junction and cell adhesion proteins within infectious centers. These morphological changes indicate activation of epithelial-to-mesenchymal transition (EMT). EMT can contribute to wound healing in respiratory epithelia by mobilizing nearby cells. Inhibiting TGF-β, and thus EMT, reduced infectious center detachment. Compared with uninfected cells, MeV-infected cells also expressed increased levels of sphingosine kinase 1 (SK1), a regulator of live cell extrusion. Live cell extrusion encourages cells to detach from respiratory epithelia by contracting the actomyosin of neighboring cells. Inhibition or induction of live cell extrusion impacted infectious center detachment rates. Thus, these two related pathways contributed to infectious center detachment in HAE. Detached infectious centers contained high titers of virus that may be protected from the environment, allowing the virus to live on surfaces longer and infect more hosts.IMPORTANCEMeasles virus (MeV) is an extremely contagious respiratory pathogen that continues to cause large, disruptive outbreaks each year. Here, we examine mechanisms of detachment of MeV-infected cells. MeV spreads cell-to-cell in human airway epithelial cells (HAE) to form groups of infected cells, termed "infectious centers". We reported that infectious centers ultimately detach from HAE as a unit, carrying high titers of virus. Viral particles within cells may be more protected from environmental conditions, such as ultraviolet radiation and desiccation. We identified two host pathways, epithelial-to-mesenchymal transition and live cell extrusion, that contribute to infectious center detachment. Perturbing these pathways altered the kinetics of infectious center detachment. These pathways influence one another and contribute to epithelial wound healing, suggesting that infectious center detachment may be a usurped consequence of the host's response to infection that benefits MeV by increasing its transmissibility between hosts.

Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell.

Translation errors, impaired folding or environmental stressors (e.g. infection) can all lead to an increase in the presence of misfolded proteins. These activate cellular responses to their removal, including intracellular protein degradation activities. Protein ubiquitylation is involved in two major degradation pathways, the ubiquitin-proteasome system and selective autophagy. In humans, the ubiquitin-like modifier-activating enzyme 1 (UBA1) is the primary E1 enzyme in the ubiquitin conjugation cascade. Viruses have evolved to exploit protein degradation pathways to complete their infection cycles. Zika virus (ZIKV) is an emerging orthoflavivirus causing serious neurologic disorders in neonates (congenital microcephaly) and adults (Guillain-Barré syndrome). Non-structural protein 5 (NS5), the largest and most conserved protein in the orthoflaviviruses, catalyses the synthesis and capping of new viral genomes. In addition to viral RNA replication in the cytoplasm, ZIKV NS5 is translocated into the nucleus to interfere with host antiviral responses. Here, we demonstrate that ZIKV NS5 co-immunoprecipitates with cellular UBA1. Immunofluorescence assays suggest that this interaction takes place primarily in the nucleus of an infected cell, although colocalization of both proteins is also detected in the cytosol. RNA interference-mediated depletion of UBA1 leads to reduced virus titres in the infected cells, while transient overexpression of UBA1 favours faster replication kinetics, with higher virus titres and protein levels detected. Moreover, UBA1-targeting drugs cause significant drops in virus infectivity. These results support a proviral role for UBA1 during ZIKV infection and encourage the potential use of inhibitors against this enzyme or its NS5-interacting epitopes as potential therapeutic targets.

Hemagglutinin with a polybasic cleavage site confers high virulence on H7N9 avian influenza viruses.

H7N9 avian influenza virus (AIV) first emerged in February 2013 in China, and early isolates were all low pathogenic (LP). After circulation for a few years in live poultry markets of China, LP H7N9 AIVs evolved into a highly pathogenic (HP) form in late 2016. Deduced amino acid sequence analysis of hemagglutinin (HA) gene revealed that all HP H7N9 AIVs have obtained four-amino-acid insertion at position 339-342 (H7 numbering), making the cleavage site from a monobasic motif (LP AIVs) to a polybasic form (HP AIVs). Notably, the polybasic cleavage site motifs are diversified, of which PEVPKRKRTAR↓GLF motif is prevalent. To elucidate the reasons accounting for its dominance, recombinant H7N9 virus carrying PEVPKRKRTAR↓GLF (rJT157-2) motif was generated based on LP H7N9 virus A/chicken/Eastern China/JT157/2016 (JT157). Besides, another two viruses containing PEVPKGKRTAR↓GLF (rJT157-1) and PEIPKRKRTAR↓GLF (rJT157-3) cleavage site motifs were also constructed as comparisons. We found that rJT157-2 showed better biological characterizations in vitro including replication kinetics, plaque size, thermal and acid stability. In addition, animal experiments demonstrated that rJT157-2 was more pathogenic to both chickens and mice with higher virus titers and induced more severe changes in the lungs. These results suggested that HP H7N9 viruses carrying PEVPKRKRTAR↓GLF motif in the HA cleavage site were most likely adaptive mutants during the evolution of H7N9 AIVs.

Isolation and characterization of a novel S1-gene insertion porcine epidemic diarrhea virus with low pathogenicity in newborn piglets

ABSTRACT Porcine epidemic diarrhea virus (PEDV) causes diarrhea and vomiting in piglets, leading to a mortality rate of 100%. Due to the high frequency of mutation, it is important to monitor the evolution of PEDV and develop potential vaccine candidates. In this study, two PEDV strains (ZJ2022 and ZQ2022) were identified by PCR. These strains were subsequently isolated, and their genome sequences, growth characteristics, and pathogenicity were compared. Phylogenetic and recombination analyses revealed that both strains belonged to GIIa-subgroup, and ZQ2022 was identified as a recombinant strain derived from ZJ2022. Further sequence analysis showed that the ZJ2022 strain had a modified top region of the S1 protein due to a three amino acid insertion (T380_Y380insGGE) in the S1 gene. According to the virus growth curve, ZJ2022 exhibited better cellular adaptation than ZQ2022, with higher viral titers from 8 hpi to 24 hpi. Additionally, ZQ2022 exhibited a high level of pathogenicity, causing severe diarrhea in piglets at 36 hpi and a 100% mortality rate by 96 hpi. In contrast, ZJ2022 showed lower pathogenicity, inducing severe diarrhea in piglets at 60 hpi, with a mortality rate of 60% at 96 hpi and 100% at 120 hpi. In summary, our findings provided evidence of the undergoing mutations in Chinese PEDV strains. Furthermore, the S gene insertion strain ZJ2022 exhibited strong cellular adaptability and low pathogenicity, making it a potential candidate strain for vaccine development.

Development of peptone-based serum-free media to support Vero CCL-81 cell proliferation and optimize SARS-CoV2 viral production.

Developing an optimal media for Vero cell lines is crucial as it directly influences cell survival, proliferation, and virus production. The use of serum in cell culture raises safety concerns in biological production. The United States Food and Drug Administration (FDA) and the European Medicines Agency have implemented stricter regulations on the use of animal-derived components in commercial protein manufacturing to ensure patient safety. This study aims to develop new chemically defined media using peptone hydrolysate as a serum-free alternative. A rational experimental design was employed to screen and optimize potential serum substitutes. Six types of peptones were tested at varying concentrations to determine the top three candidates. These candidates were subsequently combined into five mixtures to identify the two most effective serum-free media for Vero CCL-81cells. The evaluation criteria included morphology observation and proliferation assay. The two most promising serum-free formulations were further evaluated for their ability to support virus production and using TCID50 and absolute quantification by RT-qPCR. The results of this study indicate that Mixture 4, consisting of 0.5g/L Phytone™ Peptone, 0.5g/L Difco™ Soytone, and 2g/L Bacto™ Malt, significantly enhanced Vero CCL-81cell proliferation and produced high viral titers. The developed medium demonstrated comparable performance to commercially available serum-free media. Peptone-based serum-free media provide a robust alternative for supporting Vero CCL-81cell growth and are suitable for virus production.

The consequences of SARS-CoV-2 within-host persistence.

SARS-CoV-2 causes an acute respiratory tract infection that resolves in most people in less than a month. Yet some people with severely weakened immune systems fail to clear the virus, leading to persistent infections with high viral titres in the respiratory tract. In a subset of cases, persistent SARS-CoV-2 replication results in an accelerated accumulation of adaptive mutations that confer escape from neutralizing antibodies and enhance cellular infection. This may lead to the evolution of extensively mutated SARS-CoV-2 variants and introduce an element of chance into the timing of variant evolution, as variant formation may depend on evolution in a single person. Whether long COVID is also caused by persistenceof replicating SARS-CoV-2 is controversial. One line of evidence is detection of SARS-CoV-2 RNA and proteins in different body compartments long after SARS-CoV-2 infection has cleared from the upper respiratory tract. However, thus far, no replication competent virus has been cultured from individuals withlong COVIDwho are immunocompetent. In this Review, we consider mechanisms of viral persistence, intra-host evolution in persistent infections, the connection of persistent infections with SARS-CoV-2 variants and the possible role of SARS-CoV-2 persistence in long COVID. Understanding persistent infections may therefore resolve much of what is still unclear in COVID-19 pathophysiology, with possible implications for other emerging viruses.

Australian Culex annulirostris mosquitoes are competent vectors for Japanese encephalitis virus genotype IV

ABSTRACT Japanese encephalitis virus (JEV) is transmitted by Culex species of mosquitoes. In 2022, JEV belonging to a previously unrecognized lineage of genotype IV (GIV) caused a major outbreak of JE in South-eastern Australia, resulting in human cases and affecting piggeries. Cx. annulirostris has previously been implicated as the major vector of JEV in northern Australia where the virus has circulated since its first detection in 1995. Here, we showed that experimental infection of a laboratory colony of Australian Cx. annulirostris with the isolate JEV NSW/22 resulted in a 100% mosquito infection rate, with 87% of mosquito saliva samples testing positive by RT-qPCR at 14 days post-infection. Immunohistochemistry confirmed the presence of a replicating virus in the mosquito midgut and dissemination throughout the body, including the salivary glands. Our results also showed evidence of transovarial transmission of this virus; however, transstadial transmission from the eggs to the adult stage was not found. Comparison with an Indonesian isolate of GIV JEV and previous Australian isolates belonging to genotypes I and II showed that infection with JEV NSW/22 resulted in higher viral titres in the early stage of infection and higher proportions of mosquitoes with JEV-positive saliva, indicating a greater transmission potential compared to other isolates. This study provides compelling experimental evidence that Australian Cx. annulirostris is a highly efficient vector for the 2022 Australian JEV GIV outbreak strain.

Sustained applicability of SARS-CoV-2 variants identification by Sanger Sequencing Strategy on emerging various SARS-CoV-2 Omicron variants in Hiroshima, Japan

BackgroundThe evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persists, giving rise to new variants characterized by mutations in the spike protein. However, public data regarding the virus's evolutionary trend is not widely available after the downgrade of coronavirus disease 2019(COVID-19). Therefore, this study aimed to investigate the applicability of an in-house Sanger-based method for identifying SARS-CoV-2 variants, particularly focusing on newly emerged Omicron variants, and updating the epidemiology of COVID-19 during the 8th wave in Hiroshima Prefecture.ResultsA total of 639 saliva samples of individuals who had tested positive for COVID-19, received from Hiroshima City Medical Association Clinical Laboratory Center between February 01, 2023, and March 12, 2024, were included in the study. SARS-CoV-2 variants were identified in 69.3% (443/639) with the mean viral titer 2 × 106 copies/mL, and high viral titer in Omicron variant XBC.1.6* (5 × 108 copies/mL) using RT-qPCR. By partial Spike gene-based sequencing using the Sanger Sequencing strategy, Omicron sub-lineages XXB.1, BA.5, and EG.1 were identified during different periods. A comprehensive phylogenetic analysis of 7383 SARS-CoV-2 strains retrieved from GISAID, collected in Hiroshima from the onset of the COVID-19 pandemic in early 2020 until July 2024, revealed the dynamic evolution of SARS-CoV-2 variants over time. The study found a similar pattern of variant distribution between the full genomes from GISAID, and the partial genomes obtained from our screening strategy during the same period.ConclusionsOur study revealed that all SARS-CoV-2 viruses circulated in Hiroshima were Omicron variants and their sub-lineages during the 8th wave outbreak in Hiroshima. Persistent molecular surveillance of SARS-CoV-2 is needed for the decision-making and strategic planning of the public promptly. Our study added evidence for the usefulness of SARS-CoV-2 spike gene partial sequencing-based SARS-CoV-2 variant identification strategy for mass screening and molecular surveillance even though the evolution of newly emerged various SARS-CoV-2 Omicron variants.

Variable effects of transient Wolbachia infections on alphaviruses in Aedes aegypti.

Wolbachia pipientis (= Wolbachia) has promise as a tool to suppress virus transmission by Aedes aegypti mosquitoes. However, Wolbachia can have variable effects on mosquito-borne viruses. This variation remains poorly characterized, yet the multimodal effects of Wolbachia on diverse pathogens could have important implications for public health. Here, we examine the effects of transient somatic infection with two strains of Wolbachia (wAlbB and wMel) on the alphaviruses Sindbis virus (SINV), O'nyong-nyong virus (ONNV), and Mayaro virus (MAYV) in Ae. aegypti. We found variable effects of Wolbachia including enhancement and suppression of viral infections, with some effects depending on Wolbachia strain. Both wAlbB- and wMel-infected mosquitoes showed enhancement of SINV infection rates one week post-infection, with wAlbB-infected mosquitoes also having higher viral titers than controls. Infection rates with ONNV were low across all treatments and no significant effects of Wolbachia were observed. The effects of Wolbachia on MAYV infections were strikingly strain-specific; wMel strongly blocked MAYV infections and suppressed viral titers, while wAlbB had more modest effects. The variable effects of Wolbachia on vector competence underscore the importance of further research into how this bacterium impacts the virome of wild mosquitoes including the emergent human pathogens they transmit.

Lassa virus persistence with high viral titers following experimental infection in its natural reservoir host, Mastomys natalensis

Lassa virus (LASV) outbreaks in West Africa pose a significant public health threat. We investigated the infection phenotype and transmission (horizontal and vertical) of LASV strain Ba366 in its natural host, Mastomys natalensis. Here we analyze viral RNA levels in body fluids, virus titers in organs and antibody presence in blood. In adults and 2-week-old animals, LASV causes transient infections with subsequent seroconversion. However, mice younger than two weeks exhibit persistent infections lasting up to 16 months despite antibody presence. LASV can be detected in various body fluids, organs, and cell types, primarily in lung, kidney, and gonadal epithelial cells. Despite the systemic virus presence, no pathological alterations in organs are observed. Infected animals efficiently transmit the virus throughout their lives. Our findings underscore the crucial role of persistently infected individuals, particularly infected females and their progeny, in LASV dissemination within the host population.

Defense strategies against sweet potato chlorotic stunt and pakakuy virus coinfection unraveled

AbstractThis study into the response of two Ipomoea batatas (L.) Lam cultivars, Melinda and Tio Joe, to coinfection with sweet potato chlorotic stunt virus (SPCSV) and sweet potato pakakuy virus (SPPV), employed a comprehensive approach encompassing symptomatology, real‐time quantitative polymerase chain reaction, metagenomics, and transcriptomics. SPCSV is a quarantine virus with synergistic effects, which decimate yields. SPPV is the most prevalent DNA virus in sweet potato germplasm, with a tendency to persist in meristems, posing a significant risk for germplasm transfer between territories. Graft inoculation experiments revealed that while Tio Joe remained asymptomatic for 12 weeks and suppressed virus replication, Melinda displayed symptoms early on and exhibited high virus titers. Metagenomic analyses corroborated these observations and confirmed that SPCSV and SPPV were responsible. Transcriptomic analysis unveiled disparities in gene expression between Melinda and Tio Joe. Differential gene expression was heightened and altered in Melinda as the viruses disrupted its gene expression. Its defense strategies, such as inducing abscisic acid signaling, were insufficient to overcome disruptive viral effects like oxidative stress, rendering it susceptible. In contrast, Tio Joe had relatively stable differential gene expression, indicating resistance to SPPV–SPCSV coinfection. Overexpressed genes such as sirtuin, rapid alkalization factor, and nuclear pore anchor triggered quantitative resistance, supported with enriched organelles such as mitochondrion and pathways such as proteasome and cutin, suberine, and wax biosynthesis. Tio Joe maintained its genome integrity and inhibited viral replication by tightly controlling gene expression and preventing reactive oxygen species accumulation.

Omicron XBB.1.5 subvariant causes severe pulmonary disease in K18-hACE-2 mice.

Owing to their continuous evolution, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) display disparate pathogenicity in mouse models. Omicron and its sublineages have been dominant worldwide. Compared to pre-Omicron VOCs, early Omicron subvariants reportedly cause attenuated disease in human ACE-2-expressing mice (K18-hACE-2). In late 2022, the frequency of Omicron subvariant XBB.1.5 rapidly increased and it progressively replaced other circulating strains. The emergence of new strains requires current SARS-CoV-2 clinical animal model re-evaluation. In this study, we aim to characterize XBB.1.5 pathogenesis in K18-hACE-2. Herein, we demonstrated that XBB.1.5 infection is associated with significant weight loss, severe lung pathology, and substantial mortality. Intranasal XBB.1.5 infection resulted in 100% mortality in K18-hACE2 mice. High virus titers were detected in the lungs on days 3 and 5 after infection. Moreover, XBB.1.5 productively infected the cells within the nasal turbinate, olfactory bulb, intestines, and kidneys. In addition, in a subset of infected mice, we detected high virus titers in the brain. Consistently, we detected high viral antigen expression in the lungs. Furthermore, we observed severe lung injury hallmarks (e.g., immune cell infiltration, perivascular cuffing, and alveolar consolidation). Using immunofluorescence labeling and cytometric analysis, we revealed that XBB.1.5 infection leads to CD45+ cell influx into the lung parenchyma. We further demonstrated that most immune infiltrates are CD11b+ CD11c+ dendritic cells. Additionally, we detected significant induction of proinflammatory cytokines and chemokines in infected lungs. Taken together, our data show that Omicron subvariant XBB.1.5 is highly pathogenic in K18-hACE2 mice.

A single amino acid substitution in the Borna disease virus glycoprotein enhances the infectivity titer of vesicular stomatitis virus pseudotyped virus by altering membrane fusion activity.

Borna disease virus 1 (BoDV-1) causes acute fatal encephalitis in mammals, including humans. Despite its importance, research on BoDV-1 cell entry has been hindered by low infectious viral particle production in cells and the lack of cytopathic effects, which are typically useful for screening. To address these issues, we developed a method to efficiently produce vesicular stomatitis virus (VSV) pseudotyped with glycoprotein (G) of members of the genus Orthobornavirus, including BoDV-1. We discovered that optimal G expression is required to obtain a high infectivity titer of the VSV pseudotyped virus. Remarkably, the infectivity of the VSV pseudotyped virus with G from the BoDV-1 strain huP2br was significantly higher than that of the VSV pseudotyped virus with G from the He/80 strain. Mutational analysis demonstrated that the methionine at BoDV-1-G residue 307 increases the infectivity titer of VSV pseudotyped with BoDV-1-G (VSV-BoDV-1-G). A cell‒cell fusion assay indicated that this residue plays a pivotal role in membrane fusion, thus suggesting that high membrane fusion activity and a broad pH range for membrane fusion are crucial for achieving a high infectivity titer of VSV-BoDV-1-G. This finding may be extended to increase the infectivity titer of VSV pseudotyped virus with other orthobornavirus G. Our study also contributes to identifying functional domains of BoDV-1-G and provides insight into G-mediated cell entry.

THE USE OF EMBRYONATED CHICKEN EGGS WITH MATERNAL ANTIBODY TO NEWCASTLE DISEASE VIRUS FOR THE PRODUCTION OF LA SOTA STRAIN VIRAL ANTIGEN

Embryonated eggs from Specific Pathogen Free (SPF) flocks are used ideally in the production of Newcastle disease vaccine. However, in developing countries where SPF flocks are not available, embryonated eggs from healthy chicken flocks are utilized. The present study assessed the use of embryonated eggs having maternal antibodies to Newcastle disease virus (NDV) for the propagation of NDV La Sota vaccine strain. Log, hemagglutination inhibition (HI) antibody titers to NDV in sera of 18 Isa White breeder chickens were determined. Subsequently within one week, 60 eggs were collected from the breeder hens and incubated for 12 days. Maternal antibodies to NDV in the 12-day-old embrayonated eggs were determined from the egg yolk and allantoic fluid. Two fertile eggs each from five hens were then inoculated with 0.1 or 0.2 mL of La Sota vaccine reconstituted in PBS with pH of 7.4 (200 doses/mL). In addition, three eggs from different hens were inoculated with 0.1 mL of the La Sota vaccine. Log: hemagglutination (HA) NDV titers in allantoic fluid were determined from the eggs three days post inoculation. The geometric mean of HI log, antibody titers to NDV detected in sera, yolk and allantoic fluid of eggs were, 7.37, 7.09 and 1.93, respectively. There was no statistically significant difference (p 0.05) between NDV HA log: titers in allantoic fluid harvested following inoculation with 0.1 (between 9 log: to 11 log:) and 0.2 (between 8 log: to 12 log:) mL La Sota vaccine. There was no correlation (r=-0.28) between antibodies to NDV in sera of breeders and the viral titers obtained in allantoic fluid of their embryonated eggs post inoculation with La Sota vaccine. The results of this study indicate that embryonated eggs with maternal antibodies to NDV could be used for propagation of NDV to yield high viral titers. Hence, it is recommended that breeder flocks raised for the purpose of producing eggs intended for NDV vaccine production could be duly vaccinated against field NDV infection.