First characterization of major histocompatibility complex class II DRB3 diversity in cattle breeds raised in Egypt.

Genes encoding bovine leukocyte antigen (BoLA) enable the immune system to identify pathogens. Therefore, these genes have been used as genetic markers for infectious and autoimmune diseases as well as for immunological traits in cattle. Although BoLA polymorphisms have been reported in various cattle breeds worldwide, they have not been studied in cattle populations. In this study, we characterized BoLA-DRB3 in two local Egyptian populations and one foreign population using polymerase chain reaction-sequence-based typing (PCR-SBT) method. Fifty-four previously reported BoLA-DRB3 alleles and eight new alleles (BoLA-DRB3*005:08, *015:07, *016:03, *017:04, *020:02:02, *021:03, *164:01, and *165:01) were identified. Alignment analysis of the eight new alleles revealed 89.5-98.7 %, and 81.0-97.5 % nucleotide and amino acid identities, respectively, with the BoLA-DRB3 cDNA clone NR-1. Interestingly, BoLA-DRB3 in Egyptian cattle showed a high degree of allelic diversity in native (na = 28, hE > 0.95), mixed (na = 61, hE > 0.96), and Holstein (na = 18, hE > 0.88) populations. BoLA-DRB3*002:01 (14.3 %), BoLA-DRB3*001:01 (8.5 %), and BoLA-DRB3*015:01 (20.2 %) were the most frequent alleles in native, mixed, and Holstein populations, respectively, indicating that the genetic profiles differed in each population. Based on the allele frequencies of BoLA-DRB3, genetic variation among Egyptian, Asian, African, and American breeds was examined using Nei's distances and principal component analysis. The results suggested that native and mixed cattle populations were most closely associated with African breeds in terms of their gene pool, whereas Holstein cattle were more distinct from the other breeds and were closely related to Holstein cattle populations from other countries.

BoLA-DRB3 Polymorphism Associated with Bovine Leukemia Virus Infection and Proviral Load in Holstein Cattle in Egypt.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, the most prevalent neoplastic disease of cattle worldwide. The immune response to BLV and disease susceptibility and resistance in cattle are strongly correlated with the bovine leukocyte antigen (BoLA)-DRB3 allelic polymorphism. BLV infection continues to spread in Egypt, in part because the relationships between BLV infection, proviral load in Egypt, and BoLA-DRB3 polymorphism are unknown. Here, we identified 18 previously reported alleles in 121 Holstein cows using a polymerase chain reaction sequence-based typing method. Furthermore, BoLA-DRB3 gene polymorphisms in these animals were investigated for their influence on viral infection. BoLA-DRB3*015:01 and BoLA-DRB3*010:01 were identified as susceptible and resistant alleles, respectively, for BLV infection in the tested Holsteins. In addition, BoLA-DRB3*012:01 was associated with low PVL in previous reports but high PVL in Holstein cattle in Egypt. This study is the first to demonstrate that the BoLA-DRB3 polymorphism confers resistance and susceptibility to PVL and infections of BLV in Holstein cattle in Egypt. Our results can be useful for the disease control and eradication of BLV through genetic selection.

TiO2-Photocatalyst-Induced Degradation of Dog and Cat Allergens under Wet and Dry Conditions Causes a Loss in Their Allergenicity.

Allergies to dogs and cats can cause enormous damage to human health and the economy. Dog and cat allergens are mainly found in dog and cat dander and are present in small particles in the air and in carpets in homes with dogs and cats. Cleaning houses and washing pets are the main methods for reducing allergens in homes; however, it is difficult to eliminate them completely. Therefore, we aimed to investigate whether a TiO2 photocatalyst could degrade dog and cat allergens. Under wet conditions, exposure to the TiO2 photocatalyst for 24 h degraded Can f1, which is a major dog allergen extracted from dog dander, by 98.3%, and Fel d1, which is a major cat allergen extracted from cat dander, by 93.6-94.4%. Furthermore, under dry conditions, the TiO2 photocatalyst degraded Can f1 and Fel d1 by 92.8% and 59.2-68.4%, respectively. The TiO2 photocatalyst abolished the binding of dog and cat allergens to human IgE by 104.6% and 108.6%, respectively. The results indicated that the TiO2 photocatalyst degraded dog and cat allergens, causing a loss in their allergenicity. Our results suggest that TiO2 photocatalysis can be useful for removing indoor pet allergens and improving the partnership between humans and pets.

Antigenicity of subregions of recombinant bovine leukemia virus (BLV) glycoprotein gp51 for antibody detection.

Bovine leukemia virus (BLV) is an enveloped virus, found worldwide that can infect cattle and induce many subclinical symptoms and malignant tumors. BLV infection causes severe economic losses in the cattle industry. The identification of BLV-infected cattle for segregation or elimination would be the most effective way to halt the spread of BLV infection on farms, owing to the lack of effective treatments and vaccines. Therefore, antibody detection against the viral glycoprotein gp51 is an effective method for diagnosing BLV-infected animals. In this study, ten different subregions of gp51 containing a common B cell epitope are vital for developing antigens as epitope-driven vaccine design and immunological assays. Such antigens were produced in Escherichia coli expression system to react with antibodies in the serum from BLV-infected cattle and compete for antigenicity. Recombinant His-gp5156-110 and gp5133-301(full) had the same sensitivity in BLV-positive sera, indicating that antibodies responded to the limited subregion of viral gp51, a common B cell epitope. This finding provides significant information for antigen selection in BLV to use in antibody detection assays. Further studies are needed to evaluate the antigenicity of His-gp5156-110 and gp5133-301(full) as antigens for antibody detection assays using a larger number of bovine serum samples.

Anti-BLV antibodies in whey correlate with bovine leukemia virus disease progression and BoLA-DRB3 polymorphism.

Introduction: Bovine leukemia virus (BLV) belongs to the family Retroviridae and is a causative agent for enzootic bovine leucosis, the most common neoplastic disease affecting cattle worldwide. BLV proviral load (PVL) is associated with disease progression and transmission risk but requires blood collection and quantitative PCR testing. Anti-BLV antibodies in whey have been used as a diagnostic tool for BLV infection; however, quantitative utilization has not been fully investigated. Furthermore, bovine leukocyte antigen (BoLA)-DRB3 is a polymorphic gene associated with BLV infectivity and PVL, but its effect on anti-BLV antibody levels in whey from BLV infected dams is unknown. Therefore, we aimed to investigate whether it is possible to correctly predict PVL in the blood and milk based on the amount of anti-BLV antibodies in milk, and whether the BoLA-DRB3 alleles associate with the amount of anti-BLV antibodies in milk. Methods: We examined whey from 442 dams from 11 different dairy farms located in 6 prefectures in Japan, including susceptible dams carrying at least one BoLA-DRB3* 012:01 or * 015:01 allele related with high PVL, resistant dams carrying at least one BoLA-DRB3 * 002:01, * 009:02, or * 014:01:01 allele related with low PVL, and neutral dams carrying other alleles. Results: First, our results provided compelling evidence that anti-BLV antibody levels in whey were positively correlated with the anti-BLV antibody levels in serum and with BLV PVL in blood and milk, indicating the possibility of estimating BLV PVL in blood and milk by measuring anti-BLV antibody levels in whey. Thus, our results showed that antibody titers in milk might be effective for estimating BLV transmission risk and disease progression in the field. Second, we demonstrated that anti-BLV antibody levels in whey from BLV resistant dams were significantly lower than those from susceptible and neutral dams. Discussion: This is the first report suggesting that the BoLA-DRB3 polymorphism affects anti-BLV antibody levels in whey from BLV-infected dams. Taken together, our results suggested that anti-BLV antibody levels in whey, measured by enzyme-linked immunosorbent assay, may be a useful marker to diagnose the risk of BLV infection and estimate PVL in blood and milk.

Comparison of the inactivation capacity of various UV wavelengths on SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a worldwide pandemic. Ultraviolet (UV) is regarded as a very powerful tool against SARS-CoV-2. However, the inactivating effects of different UV wavelengths on SARS-CoV-2 under the same conditions have hardly been compared. Here, we showed that SARS-CoV-2 cultured in Dulbecco's modified Eagle's medium and 2% fetal bovine serum was efficiently inactivated by irradiation with 222, 254, and 265 wavelengths UV, but not at 308 nm. In addition, it was revealed that UV absorption by DMEM-2% FBS is very efficient at 222 nm. Our results present potentially important information for selecting the optimum UV wavelength according to the application.

Epigallocatechin Gallate Stabilized by Cyclodextrin Inactivates Influenza Virus and Human Coronavirus 229E.

Natural products are attractive antiviral agents because they are environment-friendly and mostly harmless. Epigallocatechin gallate (EGCg), a type of catechin, is a well-known natural antiviral agent that can inhibit various viruses. However, EGCg easily oxidizes and loses its physiological activity. Although this problem can be overcome by combining EGCg with cyclodextrin (CD-EGCg), which makes it stable in water at high concentrations, the antiviral effect of this compound remains unclear. Here, we show that in Madin-Darby canine kidney (MDCK) and MRC-5 cells, CD-EGCg is cytotoxic for 50% of cells at 85.61 and 65.34 ppm, respectively. Furthermore, CD-EGCg mainly shows its antiviral effect during the adsorption step for all four influenza virus strains (median effect concentration (EC50) was 0.93 to 2.78 ppm). Its antiviral effect post-adsorption is less intense, and no inhibitory effect is observed on influenza viruses pre-adsorption. Moreover, human coronavirus 229E (HCoV-229E) was inhibited at the adsorption step in short contact (EC50 = 2.5 ppm) and long contact conditions (EC50 = 0.5 ppm) by mixing CD-EGCg with HCoV-229E. These results suggest that CD-EGCg effectively inhibits various viruses that require an adsorption step, and is an effective tool for preventing infection.

WO3 Photocatalyst Containing Copper Inactivates SARS-CoV-2 Pango Lineage A and Omicron BA.2 Variant in Visible Light and in Darkness.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019, which has been a global pandemic. Since SARS-CoV-2 is transmitted through contaminated surfaces and aerosols, environmental disinfection is important to block the spread of the virus. Photocatalysts are attractive tools for virus inactivation and are widely used as air purifiers and coating materials. However, photocatalysts are inactive in the dark, and some of them need to be excited with light of a specific wavelength. Therefore, photocatalysts that can effectively inactivate SARS-CoV-2 in indoor environments are needed. Here, we show that a WO3 photocatalyst containing copper inactivated the SARS-CoV-2 WK-521 strain (Pango lineage A) upon irradiation with white light in a time- and concentration-dependent manner. Additionally, this photocatalyst also inactivated SARS-CoV-2 in dark conditions due to the antiviral effect of copper. Furthermore, this photocatalyst inactivated not only the WK-521 strain but also the Omicron variant BA.2. These results indicate that the WO3 photocatalyst containing copper can inactivate indoor SARS-CoV-2 regardless of the variant, in visible light or darkness, making it an effective tool for controlling the spread of SARS-CoV-2.

A Novel Class of HIV-1 Inhibitors Targeting the Vpr-Induced G2-Arrest in Macrophages by New Yeast- and Cell-Based High-Throughput Screening.

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, arrests the cell cycle of the G2 phase, and this Vpr-mediated G2 arrest is implicated in an efficient HIV-1 spread in monocyte-derived macrophages. Here, we screened new candidates for Vpr-targeting HIV-1 inhibitors by using fission yeast- and mammalian cell-based high-throughput screening. First, fission yeast strains expressing the HIV-1 Vpr protein were generated and then treated for 48 h with 20 µM of a synthetic library, including 140,000 chemical compounds. We identified 268 compounds that recovered the growth of Vpr-overexpressing yeast. The selected compounds were then tested in mammalian cells, and those displaying high cytotoxicity were excluded from further cell cycle analysis and imaging-based screening. A flow cytometry analysis confirmed that seven compounds recovered from the Vpr-induced G2 arrest. The cell toxicity and inhibitory effect of HIV-1 replication in human monocyte-derived macrophages (MDM) were examined, and three independent structural compounds, VTD227, VTD232, and VTD263, were able to inhibit HIV-1 replication in MDM. Furthermore, we showed that VTD227, but not VTD232 and VTD263, can directly bind to Vpr. Our results indicate that three new compounds and their derivatives represent new drugs targeting HIV-1 replication and can be potentially used in clinics to improve the current antiretroviral therapy.

No evidence of bovine leukemia virus proviral DNA and antibodies in human specimens from Japan.

BACKGROUND: The potential risk and association of bovine leukemia virus (BLV) with human remains controversial as it has been reported to be both positive and negative in human breast cancer and blood samples. Therefore, establishing the presence of BLV in comprehensive human clinical samples in different geographical locations is essential. RESULT: In this study, we examined the presence of BLV proviral DNA in human blood and breast cancer tissue specimens from Japan. PCR analysis of BLV provirus in 97 Japanese human blood samples and 23 breast cancer tissues showed negative result for all samples tested using long-fragment PCR and highly-sensitive short-fragment PCR amplification. No IgG and IgM antibodies were detected in any of the 97 human serum samples using BLV gp51 and p24 indirect ELISA test. Western blot analysis also showed negative result for IgG and IgM antibodies in all tested human serum samples. CONCLUSION: Our results indicate that Japanese human specimens including 97 human blood, 23 breast cancer tissues, and 97 serum samples were negative for BLV.

BoLA-DRB3 Polymorphism Controls Proviral Load and Infectivity of Bovine Leukemia Virus (BLV) in Milk.

Bovine leukemia virus (BLV), which causes enzootic bovine leukosis, is transmitted to calves through the milk of BLV-infected dams. Bovine leukocyte antigen (BoLA)-DRB3 is a polymorphic gene associated with BLV infectivity and proviral load (PVL). However, the effect of BoLA-DRB3 polymorphism on the infectivity and PVL of milk from BLV-infected dams remains unknown. This study examined milk from 259 BLV-infected dams, including susceptible dams carrying at least one BoLA-DRB3*012:01 or *015:01 allele with high PVL, resistant dams carrying at least one BoLA-DRB3*002:01, *009:02, or *014:01:01 allele with low PVL, and neutral dams carrying other alleles. The detection rate of BLV provirus and PVL were significantly higher in milk from susceptible dams than in that from resistant dams. This result was confirmed in a three-year follow-up study in which milk from susceptible dams showed a higher BLV provirus detection rate over a longer period than that from resistant dams. The visualization of infectivity of milk cells using a luminescence syncytium induction assay showed that the infectious risk of milk from BLV-infected dams was markedly high for susceptible dams compared to resistant ones. This is the first report confirming that BoLA-DRB3 polymorphism affects the PVL and infectivity of milk from BLV-infected dams.

Huntingtin-Interacting Protein 1 Promotes Vpr-Induced G2 Arrest and HIV-1 Infection in Macrophages.

Human immunodeficiency virus type 1 (HIV-1) modulates the host cell cycle. The HIV-1 accessory protein Vpr arrests the cell cycle at the G2 phase in dividing cells, and the ability of Vpr to induce G2 arrest is well conserved among primate lentiviruses. Additionally, Vpr-mediated G2 arrest likely correlates with enhanced HIV-1 infection in monocyte-derived macrophages. Here, we screened small-interfering RNA to reveal candidates that suppress Vpr-induced G2 arrest and identified Huntingtin-interacting protein 1 (HIP1) required for efficient G2 arrest. Interestingly, HIP1 was not essential for Vpr-induced DNA double-strand breaks, which are required for activation of the DNA-damage checkpoint and G2 arrest. Furthermore, HIP1 knockdown suppressed HIV-1 infection in monocyte-derived macrophages. This study identifies HIP1 as a factor promoting Vpr-induced G2 arrest and HIV-1 infection in macrophages.

Continuous monitoring system for safe managements of CO2 storage and geothermal reservoirs.

We have developed a new continuous monitoring system based on small seismic sources and distributed acoustic sensing (DAS). The source system generates continuous waveforms with a wide frequency range. Because the signal timing is accurately controlled, stacking the continuous waveforms enhances the signal-to-noise ratio, allowing the use of a small seismic source to monitor extensive areas (multi-reservoir). Our field experiments demonstrated that the monitoring signal was detected at a distance of ~ 80 km, and temporal variations of the monitoring signal (i.e., seismic velocity) were identified with an error of < 0.01%. Through the monitoring, we identified pore pressure variations due to geothermal operations and rains. When we used seafloor cable for DAS measurements, we identified the monitoring signals at > 10 km far from the source in high-spatial resolution. This study demonstrates that multi-reservoir in an extensive area can be continuously monitored at a relatively low cost by combining our seismic source and DAS.

UVC disinfects SARS-CoV-2 by induction of viral genome damage without apparent effects on viral morphology and proteins.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a pandemic threat worldwide and causes severe health and economic burdens. Contaminated environments, such as personal items and room surfaces, are considered to have virus transmission potential. Ultraviolet C (UVC) light has demonstrated germicidal ability and removes environmental contamination. UVC has inactivated SARS-CoV-2; however, the underlying mechanisms are not clear. It was confirmed here that UVC 253.7 nm, with a dose of 500 µW/cm2, completely inactivated SARS-CoV-2 in a time-dependent manner and reduced virus infectivity by 10-4.9-fold within 30 s. Immunoblotting analysis for viral spike and nucleocapsid proteins showed that UVC treatment did not damage viral proteins. The viral particle morphology remained intact even when the virus completely lost infectivity after UVC irradiation, as observed by transmission electronic microscopy. In contrast, UVC irradiation-induced genome damage was identified using the newly developed long reverse-transcription quantitative-polymerase chain reaction (RT-qPCR) assay, but not conventional RT-qPCR. The six developed long RT-PCR assays that covered the full-length viral genome clearly indicated a negative correlation between virus infectivity and UVC irradiation-induced genome damage (R2 ranging from 0.75 to 0.96). Altogether, these results provide evidence that UVC inactivates SARS-CoV-2 through the induction of viral genome damage.

SARS-CoV-2 Disinfection of Air and Surface Contamination by TiO2 Photocatalyst-Mediated Damage to Viral Morphology, RNA, and Protein.

SARS-CoV-2 is the causative agent of COVID-19, which is a global pandemic. SARS-CoV-2 is transmitted rapidly via contaminated surfaces and aerosols, emphasizing the importance of environmental disinfection to block the spread of virus. Ultraviolet C radiation and chemical compounds are effective for SARS-CoV-2 disinfection, but can only be applied in the absence of humans due to their toxicities. Therefore, development of disinfectants that can be applied in working spaces without evacuating people is needed. Here we showed that TiO2-mediated photocatalytic reaction inactivates SARS-CoV-2 in a time-dependent manner and decreases its infectivity by 99.9% after 20 min and 120 min of treatment in aerosol and liquid, respectively. The mechanistic effects of TiO2 photocatalyst on SARS-CoV-2 virion included decreased total observed virion count, increased virion size, and reduced particle surface spike structure, as determined by transmission electron microscopy. Damage to viral proteins and genome was further confirmed by western blotting and RT-qPCR, respectively. The multi-antiviral effects of TiO2-mediated photocatalytic reaction implies universal disinfection potential for different infectious agents. Notably, TiO2 has no adverse effects on human health, and therefore, TiO2-induced photocatalytic reaction is suitable for disinfection of SARS-CoV-2 and other emerging infectious disease-causing agents in human habitation.

PRMT5 Is Required for Bovine Leukemia Virus Infection In Vivo and Regulates BLV Gene Expression, Syncytium Formation, and Glycosylation In Vitro.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle and is closely related to human T-cell leukemia viruses. We investigated the role of a new host protein, PRMT5, in BLV infection. We found that PRMT5 is overexpressed only in BLV-infected cattle with a high proviral load, but not in those with a low proviral load. Furthermore, this upregulation continued to the lymphoma stage. PRMT5 expression was upregulated in response to experimental BLV infection; moreover, PRMT5 upregulation began in an early stage of BLV infection rather than after a long period of proviral latency. Second, siRNA-mediated PRMT5 knockdown enhanced BLV gene expression at the transcript and protein levels. Additionally, a selective small-molecule inhibitor of PRMT5 (CMP5) enhanced BLV gene expression. Interestingly, CMP5 treatment, but not siRNA knockdown, altered the gp51 glycosylation pattern and increased the molecular weight of gp51, thereby decreasing BLV-induced syncytium formation. This was supported by the observation that CMP5 treatment enhanced the formation of the complex type of N-glycan more than the high mannose type. In conclusion, PRMT5 overexpression is related to the development of BLV infection with a high proviral load and lymphoma stage and PRMT5 inhibition enhances BLV gene expression. This is the first study to investigate the role of PRMT5 in BLV infection in vivo and in vitro and to reveal a novel function for a small-molecule compound in BLV-gp51 glycosylation processing.

New evidence of bovine leukemia virus circulating in Myanmar cattle through epidemiological and molecular characterization.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide and causes serious problems for the cattle industry. In this study, we examined the prevalence of BLV infection and the distribution of BLV genotypes in cattle in the northern, central, and southern parts of Myanmar. The prevalence of BLV infection among Myanmar cattle (37.04%) in this study was markedly higher than the prevalence (9.1%) observed in our earlier study in which BLV was detected from the limited number of cattle only from a small area of Myanmar. Phylogenetic analysis of partial env-gp51 sequence of the isolated BLV strains revealed that there are at least three BLV genotypes (genotype-1, genotype-6, and genotype-10) in Myanmar, which have also been detected in the neighboring countries. We performed this study to estimate the BLV proviral load, which is a major diagnosis index for determining the virus transmission risk. The cattle of the three test regions with warm, wet, and humid climatic conditions (upper Sagaing, Yangon, and Kayin) exhibited a high mean proviral load, while cattle of three other regions with low annual rainfall and very high temperature (Mandalay, Magway, and upper Bago) exhibited a low mean proviral load. Further, the level of proviral load and the prevalence of BLV infection in Myanmar native cattle (N = 235) were lower than that in the hybrid cattle (Holstein Friesian × Myanmar native) (N = 62). We also observed that the cattle with high risk for BLV transmission, which have high proviral load, may enhance the BLV infection rate. Hence, to control BLV transmission, it is necessary to eliminate these cattle with high-risk for BLV transmission and to diagnose BLV provirus in cattle in the remaining regions/states of Myanmar sharing a boundary with neighboring countries.

Distinct MCM10 Proteasomal Degradation Profiles by Primate Lentiviruses Vpr Proteins.

Viral protein R (Vpr) is an accessory protein found in various primate lentiviruses, including human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) as well as simian immunodeficiency viruses (SIVs). Vpr modulates many processes during viral lifecycle via interaction with several of cellular targets. Previous studies showed that HIV-1 Vpr strengthened degradation of Mini-chromosome Maintenance Protein10 (MCM10) by manipulating DCAF1-Cul4-E3 ligase in proteasome-dependent pathway. However, whether Vpr from other primate lentiviruses are also associated with MCM10 degradation and the ensuing impact remain unknown. Based on phylogenetic analyses, a panel of primate lentiviruses Vpr/x covering main virus lineages was prepared. Distinct MCM10 degradation profiles were mapped and HIV-1, SIVmus and SIVrcm Vprs induced MCM10 degradation in proteasome-dependent pathway. Colocalization and interaction between MCM10 with these Vprs were also observed. Moreover, MCM10 2-7 interaction region was identified as a determinant region susceptible to degradation. However, MCM10 degradation did not alleviate DNA damage response induced by these Vpr proteins. MCM10 degradation by HIV-1 Vpr proteins was correlated with G2/M arrest, while induction of apoptosis and oligomerization formation of Vpr failed to alter MCM10 proteolysis. The current study demonstrated a distinct interplay pattern between primate lentiviruses Vpr proteins and MCM10.

Three YXXL Sequences of a Bovine Leukemia Virus Transmembrane Protein are Independently Required for Fusion Activity by Controlling Expression on the Cell Membrane.

Bovine leukemia virus (BLV), which is closely related to human T-cell leukemia viruses, is the causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle. The transmembrane subunit of the BLV envelope glycoprotein, gp30, contains three completely conserved YXXL sequences that fit an endocytic sorting motif. The two N-terminal YXXL sequences are reportedly critical for viral infection. However, their actual function in the viral life cycle remains undetermined. Here, we identified the novel roles of each YXXL sequence. Syncytia formation ability was upregulated by a single mutation of the tyrosine (Tyr) residue in any of the three YXXL sequences, indicating that each YXXL sequence is independently able to regulate the fusion event. The alteration resulted from significantly high expression of gp51 on the cell surface, thereby decreasing the amount of gp51 in early endosomes and further revealing that the three YXXL sequences are independently required for internalization of the envelope (Env) protein, following transport to the cell surface. Moreover, the 2nd and 3rd YXXL sequences contributed to Env protein incorporation into the virion by functionally distinct mechanisms. Our findings provide new insights regarding the three YXXL sequences toward the BLV viral life cycle and for developing new anti-BLV drugs.

Reciprocal complementation of bovine parainfluenza virus type 3 lacking either the membrane or fusion gene.

Two defective bovine parainfluenza virus type 3 (BPIV3) strains were generated, one lacking the membrane (M) protein gene and expressing EGFP (ΔM-EGFP) and the other lacking the fusion (F) protein gene and expressing mStrawberry (ΔF-mSB), by supplying deficient proteins in trans. When Madin-Darby bovine kidney (MDBK) cells were co-infected with ΔM-EGFP and ΔF-mSB at a multiplicity of infection (MOI) of 0.1, complemented viruses were easily obtained. Complemented viruses grew as efficiently as wild-type BPIV3 and could be passaged in MDBK cell cultures even at an MOI of 0.01, possibly due to multiploid virus particles containing genomes of both ΔM-EGFP and ΔF-mSB. This reciprocal complementation method using two defective viruses would be useful to express large or multiple proteins in cell cultures using paramyxovirus vectors.