Blind Passage Research Articles

First isolation and whole genome characterization of porcine deltacoronavirus from pigs in Peru.

Porcine deltacoronavirus is a newly emergent enteric pathogen affecting swine farms worldwide. It has been detected in several countries in Europe, Asia and North America; yet, it has not been reported in South America. In November 2019, an enteric disease outbreak in a pig farm located in San Martin, Peru, was reported along with submission of three intestinal samples from pigs who succumbed to the disease. Samples were processed for molecular detection by qRT-PCR, viral isolation and further sequencing analysis. A taqman-based RT-PCR was performed to differentiate among the most relevant swine enteric coronaviruses described to date. All samples were positive to porcine deltacoronavirus with a cycle threshold (Ct) value between 9 and 14, revealing a high viral load, while testing negative to porcine epidemic diarrhea and transmissible gastroenteritis viruses. Following detection, viral isolation was performed using PK-15 and Vero cell lines. After 5 days of inoculation, no cytopathic effect was observed. A second blind passage allowed the observation of cytopathic effect on PK-15 cells, while it remained absent in Vero cells. A fluorescence test using an anti-N monoclonal antibody confirmed viral replication. One sample was processed for whole genome sequencing (WGS). In short, raw reads were imported into CLC genomics and assembled de novo. Out of 479k reads generated from the sample, 436k assembled into a 25,501bp contig which was 99.5% identical to a reference porcine deltacoronavirus strain from the USA within the North American phylogroup. Yet, there are relevant differences at the nucleotide and amino acid levels compared with previously described porcine deltacoronavirus strains. Altogether, our findings represent the first report of porcine deltacoronavirus in South America, which provides information of its evolutionary origin. Thus, this study offers new insights into the molecular epidemiology of porcine deltacoronavirus infections in the swine industry.

First isolation and genomic characterization of bovine parechovirus from faecal samples of cattle in Japan

A novel picornavirus was isolated from the faeces of a diarrhoeic cow using MA-104 cells at the third blind passage. This virus, named Den1/2021/JPN, was completely sequenced using total RNA from the cell culture supernatant by deep sequencing. The genome of Den1/2021/JPN had a standard picornavirus genome organisation with conserved picornaviral motifs. The 5' untranslated region harboured a type-II internal ribosomal entry site. Den1/2021/JPN was most closely related to a bovine parechovirus (Bo_ParV) named cow/2018/4, which has been recently identified in publicly available databases. Phylogenetic analyses and pairwise sequence comparison revealed that Den1/2021/JPN and Bo_ParV cow/2018/4 clustered with parechoviruses and were most closely related to Parechovirus E identified in birds of prey, exhibiting nucleotide sequence similarity of 64.2-64.5 %, 58.6-59.7 % and 66.3-66.4 % in the polyprotein, P1 and 2C+3 CD coding regions, respectively. This study presents the first report on the isolation of Bo_ParV. Den1/2021/JPN and Bo_ParV cow/2018/4, which are candidates for a novel species in the genus Parechovirus.

Isolation and whole-genome sequencing of a novel aviadenovirus from owls in Japan.

Adenoviruses have been reported to infect a variety of birds. Here, we isolated a novel adenovirus from the liver of a dead owl chick (Bengal eagle owl; Bubo bengalensis) at a raptor-breeding facility in Japan and determined the complete genome sequence of the virus. We performed necropsies on the dead owl chicks and found that they had enlarged livers, pericardial edema, and focal necrosis of the liver tissue. Transmission electron microscopy of the liver tissue revealed a virus-like structure, appearing as paracrystalline arrays in the nucleus, and immunohistochemical staining with anti-adenovirus antibodies showed positive reactions in hepatocytes and other cells. Attempts to isolate the virus from homogenized liver tissue of a dead owl chick showed a cytopathic effect on chicken-derived cultured cells after multiple blind passages. Further, we determined the complete genome sequence of this virus and performed phylogenetic analysis, revealing that this adenovirus belongs to the genus Aviadenovirus, forming a cluster with fowl and turkey aviadenoviruses. The amino acid sequence divergence between the DNA polymerase of this virus and its closest known adenovirus relative is approximately 29%, implying that this virus can be assigned to a new species in the genus Aviadenovirus. Based on our data, this novel owl adenovirus is a likely cause of fatal infections in owls, which may threaten wild and captive owl populations. Further, this virus is unique among raptor adenoviruses in that it infects chicken-derived cultured cells, raising the importance of further investigations to evaluate interspecies transmission of this virus.

Isolation and Identification of a Rare Spike Gene Double-Deletion SARS-CoV-2 Variant From the Patient With High Cycle Threshold Value.

Coronavirus disease 2019 (COVID-19) is an emerging life-threatening pulmonary disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which originated in Wuhan, Hubei Province, China, in December 2019. COVID-19 develops after close contact via inhalation of respiratory droplets containing SARS-CoV-2 during talking, coughing, or sneezing by asymptomatic, presymptomatic, and symptomatic carriers. This virus evolved over time, and numerous genetic variants have been reported to have increased disease severity, mortality, and transmissibility. Variants have also developed resistance to antivirals and vaccination and can escape the immune response of humans. Reverse transcription polymerase chain reaction (RT–PCR) is the method of choice among diagnostic techniques, including nucleic acid amplification tests (NAATs), serological tests, and diagnostic imaging, such as computed tomography (CT). The limitation of RT–PCR is that it cannot distinguish fragmented RNA genomes from live transmissible viruses. Thus, SARS-CoV-2 isolation by using cell culture has been developed and makes important contributions in the field of diagnosis, development of antivirals, vaccines, and SARS-CoV-2 virology research. In this research, two SARS-CoV-2 strains were isolated from four RT–PCR-positive nasopharyngeal swabs using VERO E6 cell culture. One isolate was cultured successfully with a blind passage on day 3 post inoculation from a swab with a Ct > 35, while the cells did not develop cytopathic effects without a blind passage until day 14 post inoculation. Our results indicated that infectious SARS-CoV-2 virus particles existed, even with a Ct > 35. Cultivable viruses could provide additional consideration for releasing the patient from quarantine. The results of the whole genome sequencing and bioinformatic analysis suggested that these two isolates contain a spike 68-76del+spike 675-679del double-deletion variation. The double deletion was confirmed by amplification of the regions spanning the spike gene deletion using Sanger sequencing. Phylogenetic analysis revealed that this double-deletion variant was rare (one per million in public databases, including GenBank and GISAID). The impact of this double deletion in the spike gene on the SARS-CoV-2 virus itself as well as on cultured cells and/or humans remains to be further elucidated.

Cichlids endemic to India are not susceptible to Tilapia Lake virus infection

Infection with Tilapia tilapinevirus, also known as Tilapia Lake virus (TiLV) is responsible for high mortalities in tilapia and its hybrid species and presents a significant threat to the tilapia farming industry. Till date, TiLV infections have not been reported in other cichlids that are co-cultured with tilapia. Nonetheless, as natural host switch is a frequent phenomenon among fish viruses, the current study was undertaken to check the susceptibility of three Indian cichlid species, viz. pearlspot (Etroplus suratensis), orange chromide (Pseudetroplus maculatus), and canara pearlspot (E. canarensis), to TiLV vis-a-vis a susceptible host, Nile tilapia (Oreochromis niloticus) using in vivo and in vitro methods. The experimentally-infected Etroplus spp. did not exhibit any clinical signs of infection. Nile tilapia, on the other hand, exhibited a cumulative mortality of 75% at 12 days post challenge, with clinical signs typical of TiLV disease. Three cell lines derived from Etroplus spp. namely, orange chromide fin (OCF), canara pearlspot fin (CPSF), and pearlspot fin (PSF) cell lines did not show any cytopathic effects (CPE) even after 5 blind passages upon inoculation with TiLV-positive tissue homogenate from tilapia. CPE were apparent in the O. niloticus liver (OnlL) cell line after 72 h and the presence of TiLV was confirmed by RT PCR. Thus, the absence of clinical signs and TiLV-specific amplification in the liver and brain tissues of the experimentally infected Etroplus spp., as well as the absence of CPE and the presence of TiLV genome in the three Etroplus cell lines confirmed that Indian cichlids are not susceptible to TiLV infection.

Characterization of SARS-CoV-2 East Java isolate, Indonesia

Background: Incidents of SARS-CoV-2 in East Java increased steadily, and it became the second epicenter in Indonesia. The COVID-19 pandemic caused a dire multisectoral crisis all around the world. This study investigates and characterizes local isolates from East Java, Indonesia. Methods: There were 54 patients suspected with SARS-COV-2 infection and 27 patients were COVID-19 positive. Virus isolates were obtained from COVID-19 inpatients’ nasopharyngeal swabs at the Dr Soetomo Teaching Hospital, Surabaya. There were only three isolates (#6, #11, #35) with good growth characteristics. Serial blind passage and cytopathic effect observation in the Vero E6 cell line were performed for virus isolation. Confirmation of the SARS-CoV-2 infection was proven by means of reverse transcriptase-polymerase chain reactions using SARS-CoV-2 specific primers, scanning electron microscopy, and scanning transmission electron microscopy examination. Whole genome sequencing was performed using ARTIC protocol. Furthermore, SARS-CoV-2 characterization was identified through a western blot using rabbit serum immunized with inactive SARS-CoV-2 vaccine and human natural COVID-19 infection serum. Results: Spike gene analysis of three samples (#6, #11, #35) found that the D614G mutation was detected in all isolates, although one isolate exhibited the D215Y and E484D mutation. Based on whole genome analysis, those three isolates were included in clade 20A, and two isolates were included in lineage B.1.6 with one isolate belongs to lineage B.1.4.7. Conclusion: Based on molecular characterization and immunogenicity of SARS-CoV-2 East Java, Indonesia showed high titer and it has mutation in some regions.

Use of guanidine thiocyanate-based nucleic acid extraction buffers to inactivate poliovirus in potentially infectious materials

The efforts of the Global Poliovirus Eradication Initiative (GPEI) have brought about the near elimination of poliovirus worldwide. The World Health Organization has issued guidelines for the safe handling and containment of infectious materials (IM) and potentially infectious materials (PIM) following poliovirus eradication. Inactivation of poliovirus in IM and PIM is needed to prevent inadvertent re-introduction of polioviruses post-eradication. In this study, we investigated the use of guanidine thiocyanate-based nucleic acid extraction buffers from commercially available nucleic acid extraction kits to inactivate poliovirus in cell culture isolates and stool suspensions, two common types of poliovirus IM and PIM, respectively. Incubation with selected nucleic acid extraction buffers or extraction buffers supplemented with ethanol reduced the infectivity of high-titer wild poliovirus type 1 (WPV1), wild poliovirus type 3 (WPV3), Sabin 1 (SL1), and Sabin 3 (SL3) cell culture isolates below the limit of detection in CCID50 assays. Stool suspensions containing WPV1, WPV3, SL1, SL2, or SL3 were also inactivated by the extraction buffers tested. Blind passage of WPV1-spiked stool suspensions confirmed complete inactivation of WPV1 after incubation with extraction buffers. Moreover, treatment with a buffer consisting of 4 M guanidine thiocyanate with 30 % ethanol inactivated a high-titer WPV1 culture isolate and a WPV1-spiked stool suspension. Taken together, these results show that guanidine thiocyanate-based nucleic acid extraction buffers are an effective means of inactivating poliovirus IM and PIM, and thus will be instrumental in ensuring containment compliance and preventing potential re-emergence of contained polioviruses.

Stability of African swine fever virus on spiked spray-dried porcine plasma.

African swine fever (ASF) is a viral disease that affects members of the Suidae family. The notifiable disease is considered a major threat to the pig industry, animal health, and food security worldwide. According to the European Food Safety Authority, ASF virus (ASFV) survival and transmission in feed and feed materials is a major research gap. Against this background, the objective of this study was to determine the survival of ASFV on spiked spray-dried porcine plasma (SDPP) when stored at two different temperatures. To this means, commercial SDPP granules were contaminated with high titers of ASFV in a worst-case external contamination scenario. Three samples per time point and temperature condition were subjected to blind passaging on macrophage cultures and subsequent haemadsorption test to determine residual infectivity. In addition, viral genome was detected by real-time PCR. The results indicate that heavily contaminated SDPP stored at 4°C remains infectious for at least 5 weeks. In contrast, spiked SDPP stored at room temperature displayed a distinct ASFV titer reduction after 1 week (>2.8 log levels) and complete inactivation after 2weeks (>5.7 log levels). In conclusion, the residual risk of ASFV transmission through externally contaminated SDPP is low if SDPP is stored at room temperature (21 ± 2°C) for a period of at least 2weeks before feeding.

Characterization ofSARS-CoV-2 East Javaisolate, Indonesia.

Background:Incidentsof SARS-CoV-2 inEast Javaincreased steadily, and itbecame the second epicenter in Indonesia.TheCOVID-19 pandemic caused a dire multisectoral crisis all around the world.Thisstudyinvestigates and characterizeslocal isolates from East Java,Indonesia. Methods:There were54patients suspected with SARS-COV-2 infectionand27 patients were COVID-19 positive.Virus isolateswereobtained fromCOVID-19 inpatients'nasopharyngeal swabsat theDrSoetomoTeaching Hospital, Surabaya.There were only threeisolates(#6, #11, #35) withgoodgrowth characteristics.Serial blind passage and cytopathic effect observation intheVeroE6 cell line wereperformed for virus isolation. Confirmation ofthe SARS-CoV-2 infection was proven by means of reverse transcriptase-polymerase chain reactions using SARS-CoV-2 specific primers, scanning electron microscopy, and scanning transmission electron microscopy examination. Whole genome sequencing was performed using ARTIC protocol. Furthermore, SARS-CoV-2 characterization wasidentifiedthroughawestern blot using rabbit serum immunized with inactive SARS-CoV-2 vaccine andhuman natural COVID-19 infection serum. Results:Spike gene analysis of threesamples (#6, #11, #35)found thattheD614G mutationwasdetected inallisolates, although one isolate exhibited theD215Y and E484D mutation. Based on whole genome analysis,those three isolates were included in clade 20A, and two isolates were included in lineage B.1.6 with one isolate belongs to lineage B.1.4.7. Conclusion:Based onmolecular characterizationand immunogenicityof SARS-CoV-2 East Java, Indonesiashowed hightiter and it has mutation in some regions.

Isolation and molecular characterization of a virulent Marek's Disease virus serotype-1from Andhra Pradesh, India.

Marek's disease (MD) is one of the most significant neoplastic diseases of poultry caused by Marek's disease virus (MDV), an oncogenic avian herpesvirus which is responsible for great economic losses to the poultry industry worldwide. MD is being manifested as an acute disease with lymphomas in multiple visceral organs. In the present study, an outbreak of MD was investigated in one of the poultry farms from Andhra Pradesh, India. The gross lesions in the affected birds included lymphomas in different visceral organs like liver, spleen, proventriculus, heart and ovaries. Histopathology revealed presence of uniform lymphoblastoid cell infiltration typical of MD. The isolation of the virus was carried out in duck embryo fibroblast cells. After three blind passages, the cell cultures revealed plaque formation typical of MDV. Further confirmation of the virus was carried out by PCR targeting 132 bp repeats of serotype‑1 MDV and the oncogenes Meq and vIL‑8 were amplified and sequenced. The nucleotide and phylogenetic analysis of the virus confirmed the virus as virulent serotype‑ 1 MDV. The present outbreak suggests the need for change in the vaccination regimen of MD vaccination with appropriate serotype‑ 1 MD vaccines in Indian poultry flocks as the HVT and bivalent vaccines are unable to protect the flocks against virulent MDV.

Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cells

BackgroundAn outbreak of novel coronavirus (SARS-CoV-2)-associated respiratory infectious diseases (COVID-19) emerged in 2019 and has spread rapidly in humans around the world. The demonstration of in vitro infectiousness of respiratory...

Lack of viable severe acute respiratory coronavirus virus 2 (SARS-CoV-2) among PCR-positive air samples from hospital rooms and community isolation facilities.

Understanding the extent of aerosol-based transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important for tailoring interventions for control of the coronavirus disease 2019 (COVID-19) pandemic. Multiple studies have reported the detection of SARS-CoV-2 nucleic acid in air samples, but only one study has successfully recovered viable virus, although it is limited by its small sample size. We aimed to determine the extent of shedding of viable SARS-CoV-2 in respiratory aerosols from COVID-19 patients. In this observational air sampling study, air samples from airborne-infection isolation rooms (AIIRs) and a community isolation facility (CIF) housing COVID-19 patients were collected using a water vapor condensation method into liquid collection media. Samples were tested for presence of SARS-CoV-2 nucleic acid using quantitative real-time polymerase chain reaction (qRT-PCR), and qRT-PCR-positive samples were tested for viability using viral culture. Samples from 6 (50%) of the 12 sampling cycles in hospital rooms were positive for SARS-CoV-2 RNA, including aerosols ranging from <1 µm to >4 µm in diameter. Of 9 samples from the CIF, 1 was positive via qRT-PCR. Viral RNA concentrations ranged from 179 to 2,738 ORF1ab gene copies per cubic meter of air. Virus cultures were negative after 4 blind passages. Although SARS-CoV-2 is readily captured in aerosols, virus culture remains challenging despite optimized sampling methodologies to preserve virus viability. Further studies on aerosol-based transmission and control of SARS-CoV-2 are needed.

Isolation and characterization of lumpy skin disease virus from cattle in India.

Lumpy skin disease (LSD) has devastating economic impact. During the last decade, LSD had spread to climatically new and previously disease-free countries, which also includes its recent emergence in the Indian subcontinent (2019). This study deals with the LSD outbreak(s) from cattle in Ranchi (India). Virus was isolated from the scabs (skin lesions) in the primary goat kidney cells. Phylogenetic analysis based on nucleotide sequencing of LSD virus (LSDV) ORF011, ORF012 and ORF036 suggested that the isolated virus (LSDV/Bos taurus-tc/India/2019/Ranchi) is closely related to Kenyan LSDV strains. Further, we adapted the isolated virus in Vero cells. Infection of the isolated LSDV to Vero cells did not produce cytopathic effect (CPE) until the 4th blind passage, but upon adaptation, it produced high viral titres in the cultured cells. The kinetics of viral DNA synthesis and one-step growth curve analysis suggested that Vero cell-adapted LSDV initiates synthesizing its genome at ~24 hours post-infection (hpi) with a peak level at ~96 hpi whereas evidence of progeny virus particles was observed at 36-48 hours (h) with a peak titre at ~120 h. To the best of our knowledge, this study describes the first successful isolation of LSDV in India, besides providing insights into the life cycle Vero cell-adapted LSDV.

Isolation and whole protein characterization of species A and B bovine rotaviruses from Chinese calves.

Rotaviruses (RVs) account for severe diarrhea in children and young animals globally. In the current study, the fecal samples of diarrheic calves from a beef farm in Inner Mongolia were screened for RVA by ELISA and RT-PCR, followed by culture of three positive RVA samples in the MA-104 cell line. After 10 blind passages, cytopathic effects (CPE) appeared as detachment, granulation, and clustering of the inoculated cells. The virus isolates were identified by RT-PCR (VP6 gene RVA) and ESI-LC-MS/MS for whole protein sequencing. The protein sequences demonstrated the presence of two strains from species A rotavirus and one RVB strain; RVA/Cow-tc/CHN/35333/2019/G6P[5] was mixed with one RVB strain (RVB/Cow-tc/CHN/35334/2019/G5P[3]) in two samples, and RVA/Cow-tc/CHN/10927/2019/G8P[7] was found in one sample. They are of genotype constellations (G6-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3), (G8-P[7]-I5-R1-C1- M2-A1-N1-T1-E1-H1), and (G5-P[3]-I3-R5-C5-A5-N4-H5), respectively. Besides, phylogenetic analysis of the obtained sequences demonstrated viral evolution.

Establishment of a mouse- and egg-adapted strain for the evaluation of vaccine potency against H3N2 variant influenza virus in mice.

Sporadic spreads of swine-origin influenza H3N2 variant (H3N2v) viruses were reported in humans, resulting in 437 human infections between 2011 and 2021 in the USA. Thus, an effective vaccine is needed to better control a potential pandemic for these antigenically distinct viruses from seasonal influenza. In this study, a candidate vaccine strain with efficient growth capacity in chicken embryos was established through serial blind passaging of A/Indiana/08/2011 (H3N2)v in mice and chicken embryos. Seven amino acid substitutions (M21I in PA; A138T, N165K, and V226A in HA; S312L in NP; T167I in M1; G62A in NS1 proteins) were found in the passaged viruses without a major change in the antigenicity. This mouse- and egg-adapted virus was used as a vaccine and challenge strain in mice to evaluate the efficacy of the H3N2v vaccine in different doses. Antibodies with high neutralizing titers were induced in mice immunized with 100 µg of inactivated whole-virus particles, and those mice were significantly protected from the challenge of homologous strain. The findings indicated that the established strain in the study was useful for vaccine study in mouse models.

Isolation of peste des petits ruminants virus using primary goat kidney cell culture from kidneys obtained at slaughter.

BackgroundTraditionally isolation of peste des petits ruminant virus (PPRV) is performed in Vero cells that takes several blind passages before observing typical cytopathic effects (CPEs). As an alternate, researchers have been using lamb kidney (LK) cells but day‐old lambs are difficult to obtain and requires animal sacrifice.ObjectiveWe established a primary goat kidney (GK) cell culture from the kidneys obtained at slaughter.MethodsThe kidney of Black Bengal goats were collected from slaughter house and processed to make single cell suspension. The cells were resuspended in appropriate culture medium and maintained under optimum culture condition.ResultsThe 80% confluent monolayer of GK cells was obtained after 15–20 days post seeding. Upon infection with a field isolate of PPRV, the well‐developed CPEs characterized by cell rounding, vacuolation in the cytoplasm and fusion of cells were observed after 48 hr post infection. Virus quantification in the culture supernatant revealed more viral RNA in GK cells than LK cells. The multicycle growth analysis of PPRV showed a steady increase in the virus loads in the culture supernatant of infected GK cells, suggesting an adaptation of the PPRV in GK cells.ConclusionsThe findings suggest that primary GK cells can be successfully prepared from the mature kidney cortical tissues and can be used for the isolation of PPRV. This system could reduce the unnecessary sacrifice of lambs or kids. Since kidneys of slaughtered goats are available throughout the year, using this protocol primary cell culture from mature goat kidney can provide primary cells to the laboratory throughout the year.

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.

Validation of a binary ethylenimine (BEI) inactivation procedure for biosafety treatment of foot-and-mouth disease viruses (FMDV), vesicular stomatitis viruses (VSV), and swine vesicular disease virus (SVDV)

Binary ethylenimine (BEI) has been widely used as a virucide to inactivate viruses. For regulatory exclusion of a select agent, the United States Federal Select Agent Program (FSAP) requires an inactivation procedure that renders a select agent non-viable but allows the select agent to retain antigenic characteristics for future use must be validated, and the inactivated agent must be confirmed by a viability testing. In this curve-based validation study, we examined impacts of BEI concentration, treatment temperature, and time on our in-house inactivation procedures of Foot-and-Mouth Disease Virus (FMDV), Vesicular Stomatitis Virus (VSV), and Swine Vesicular Disease Virus (SVDV). The inactivation efficacy was confirmed by virus titration and 3 consecutive blind passages on the monolayers of susceptible cells. A linear correlation between the virus titer reduction and BEI concentration, treatment time, and temperature was established. The results confirmed our in-house BEI inactivation procedure of two doses of 1.5 mM BEI treatment at 37 °C, 1st dose for 24 h, then 2nd dose for 6 more hours for a total of 30 h BEI contact time, can ensure complete inactivation of FMDV, VSV, and SVDV.

P108 and T109 on E2 Glycoprotein Domain I Are Critical for the Adaptation of Classical Swine Fever Virus to Rabbits but Not for Virulence in Pigs.

The classical swine fever virus (CSFV) live attenuated vaccine C-strain is adaptive to rabbits and attenuated in pigs, in contrast with the highly virulent CSFV Shimen strain. Previously, we demonstrated that P108 and T109 on the E2 glycoprotein (E2P108-T109) in domain I (E2DomainI) rather than R132, S133, and D191 in domain II (E2DomainII) determine C-strain's adaptation to rabbits (ATR) (Y. Li, L. Xie, L. Zhang, X. Wang, C. Li, et al., Virology 519:197-206, 2018). However, it remains elusive whether these critical amino acids affect the ATR of the Shimen strain and virulence in pigs. In this study, three chimeric viruses harboring E2P108-T109, E2DomainI, or E2DomainII of C-strain based on the non-rabbit-adaptive Shimen mutant vSM-HCLVErns carrying the Erns glycoprotein of C-strain were generated and evaluated. We found that E2P108-T109 or E2DomainI but not E2DomainII of C-strain renders vSM-HCLVErns adaptive to rabbits, suggesting that E2P108-T109 in combination with the Erns glycoprotein (E2P108-T109-Erns) confers ATR on the Shimen strain, creating new rabbit-adaptive CSFVs. Mechanistically, E2P108-T109-Erns of C-strain mediates viral entry during infection in rabbit spleen lymphocytes, which are target cells of C-strain. Notably, pig experiments showed that E2P108-T109-Erns of C-strain does not affect virulence compared with the Shimen strain. Conversely, the substitution of E2DomainII and Erns of C-strain attenuates the Shimen strain in pigs, indicating that the molecular basis of the CSFV ATR and that of virulence in pigs do not overlap. Our findings provide new insights into the mechanism of adaptation of CSFV to rabbits and the molecular basis of CSFV adaptation and attenuation.IMPORTANCE Historically, live attenuated vaccines produced by blind passage usually undergo adaptation in cell cultures or nonsusceptible hosts and attenuation in natural hosts, with a classical example being the classical swine fever virus (CSFV) lapinized vaccine C-strain, which was developed by hundreds of passages in rabbits. However, the mechanism of viral adaptation to nonsusceptible hosts and the molecular basis for viral adaptation and attenuation remain largely unknown. In this study, we demonstrated that P108 and T109 on the E2 glycoprotein together with the Erns glycoprotein of the rabbit-adaptive C-strain confer adaptation to rabbits on the highly virulent CSFV Shimen strain by affecting viral entry during infection but do not attenuate the Shimen strain in pigs. Our results provide vital information on the different molecular bases of CSFV adaptation to rabbits and attenuation in pigs.

Myristoylation of EV71 VP4 is Essential for Infectivity and Interaction with Membrane Structure.

The Enterovirus 71 (EV71) VP4 is co-translationally linked to myristic acid at its amino-terminal glycine residue. However, the role of this myristoylation in the EV71 life cycle remains largely unknown. To investigate this issue, we developed a myristoylation-deficient virus and reporter (luciferase) pseudovirus with a Gly-to-Ala mutation (G2A) on EV71 VP4. When transfecting the EV71-G2A genome encoding plasmid in cells, the loss of myristoylation on VP4 did not affect the expression of viral proteins and the virus morphology, however, it did significantly influence viral infectivity. Further, in myristoylation-deficient reporter pseudovirus-infected cells, the luciferase activity and viral genome RNA decreased significantly as compared to that of wild type virus; however, cytopathic effect and viral capsid proteins were not detected in myristoylation-deficient virus-infected cells. Also, although myristoylation-deficient viral RNA and proteins were detected in the second blind passage of infection, they were much fewer in number compared to that of the wild type virus. The replication of genomic RNA and negative-strand viral RNA were both blocked in myristoylation-deficient viruses, suggesting that myristoylation affects viral genome RNA release from capsid to cytoplasm. Besides, loss of myristoylation on VP4 altered the distribution of VP4-green fluorescent protein protein, which disappeared from the membrane structure fraction. Finally, a liposome leakage assay showed that EV71 myristoylation mediates the permeability of the model membrane. Hence, the amino-terminal myristoylation of VP4 is pivotal to EV71 infection and capsid-membrane structure interaction. This study provides novel molecular mechanisms regarding EV71 infection and potential molecular targets for antiviral drug design.