Quantitative performance of digital ELISA for the highly sensitive quantification of viral proteins and influenza virus.

A single-molecule assay (SiMoA) using a digital enzyme-linked immunosorbent assay (ELISA) has been attracting attention as a promising method that can detect viruses with ultra-high sensitivity. However, the quantitative application of digital ELISA has not been adequately reported. Therefore, in this study, we first evaluated the linearity and sensitivity of digital ELISA using a Certified Reference Material of C-reactive protein (NMIJ CRM 6201-c) as a quality control material. Next, we originally screened those antibody pair that are suitable for detecting recombinant viral proteins of influenza A virus, nucleoprotein (NP), and hemagglutinin (HA), and established the measurement system. Under optimized conditions, the limit of detection (LOD) of NP and HA was 0.59 fM and 0.99 fM, and the coefficient of determination, R2, was 0.9998 and 0.9979, respectively. Two subtypes of influenza virus, A/Puerto Rico/8/1934 (H1N1) [PR8] and A/Panama/2007/99 (H3N2) [Pan99], were also quantified under established conditions, and the LOD of PR8 was 3.1 × 102 PFU/mL on targeting NP and 7.4 × 102 PFU/mL on targeting HA. The LOD of Pan99 was 5.3 × 102 PFU/mL on targeting NP. The specificity and robustness of the recombinant viral protein and influenza virus measurements using digital ELISA were also evaluated. Our measurement system showed enough specificity to discriminate the viral subtypes properly and showed sufficient inter- and intra-assay variations for both measurements of recombinant viral proteins and viruses, except for NP-targeting virus measurement.

Single Amino Acid Substitution in the Receptor Binding Domain of Spike Protein Is Sufficient To Convert the Neutralization Profile between Ethiopian and Middle Eastern Isolates of Middle East Respiratory Coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes MERS, which is endemic in the Middle East. The absence of human cases in Africa despite the presence of MERS-CoV suggests virological differences between MERS-CoVs in Africa and the Middle East. In fact, in the laboratory, recombinant MERS-CoV carrying the spike (S) protein of Ethiopian isolates exhibits attenuated properties, being more easily neutralized and replicating slower than viruses carrying the S protein of Middle Eastern isolate, EMC. In this study, to identify the amino acids that define the different virological features between Ethiopian and Middle Eastern MERS-CoVs, neutralization titers and viral replication were evaluated using recombinant MERS-CoVs carrying amino acid substitution(s) in the S protein. A single amino acid difference introduced into the receptor binding domain was sufficient to reverse the difference in the neutralizing properties of the S protein between Ethiopian and Middle Eastern MERS-CoVs. Furthermore, amino acid mutations in the S1 and S2 regions of S protein were collectively involved in slow viral replication. Since even a single amino acid difference in S protein can reverse the viral properties of MERS-CoV, it should be noted that multiple mutations may induce a significant change. Careful monitoring of genetic alterations in MERS-CoVs in Africa is therefore required to detect the emergence of virulent strains generated by a few genetic differences. IMPORTANCE There have been no reported cases of human Middle East respiratory syndrome (MERS) in Africa, despite the presence of MERS coronavirus (MERS-CoV). Previous studies have shown that recombinant MERS-CoV carrying the S protein of an Ethiopian isolate replicated slower and was more easily neutralized relative to MERS-CoV carrying the S protein of a Middle Eastern isolate. In this study, we investigated the amino acid(s) in S protein associated with the different viral characteristics between Ethiopian and Middle Eastern MERS-CoVs. The results revealed that a single amino acid difference in the receptor binding domain was sufficient to reverse the neutralization profile. This implies that slight genetic changes can alter the predominant population of MERS-CoV, similar to the transition of variants of severe acute respiratory syndrome coronavirus-2. Careful genetic monitoring of isolates is important to detect the spread of possible virulent MERS-CoVs generated by mutation(s).

Survey of bovine foamy virus infection among cattle in Japan and comparison with bovine leukemia virus infection.

The prevalence of bovine foamy virus (BFV) infections in cattle on farms in the Kanto region of Japan was determined using agar gel immunodiffusion (AGID) test and polymerase chain reaction (PCR). Six out of 20 farms contained BFV-positive cattle. Furthermore, 16.7% (91/545) of all cattle tested positive for BFV. This suggested that BFV-infected cattle are widely prevalent in Japan. Positive results for BFV infection were consistent between AGID and PCR tests. Additionally, we tested for bovine leukemia virus (BLV) infections at nine farms, primarily those containing BFV-infected cows. At each farm, the infection rate of BFV was lower than that of BLV. Further, cattle that were PCR-positive but antibody-negative, indicating immune tolerance to BFV, were not detected.

Evaluation of serological assays available in a biosafety level 2 laboratory and their application for survey of Middle East respiratory syndrome coronavirus among livestock in Ethiopia.

A serological survey of Middle East respiratory syndrome coronavirus (MERS-CoV) was conducted among dromedary camels and herbivorous animals sharing the same pasturage in Ethiopia. The pseudotyped vesicular stomatitis virus coated with the spike protein of MERS-CoV was used in virus neutralization (VN) tests performed in a biosafety level (BSL)-2 laboratory. The results were similar to those obtained from the VN test using live MERS-CoV and were more sensitive than the ELISA performed using synthetic MERS S1 fragment as the antigen as well as the competitive ELISA performed using a monoclonal antibody against MERS-CoV. According to the comprehensive results of the four types of serodiagnosis methods, positive antibodies were detected only in dromedary camels and the remaining herbivorous animals were not infected with the virus. Moreover, using the present procedure, serological tests for MERS-CoV can be conducted even in BSL 2 laboratory.

Middle East Respiratory Syndrome Coronavirus in Dromedaries in Ethiopia Is Antigenically Different From the Middle East Isolate EMC.

Middle East respiratory syndrome (MERS) is an emerging respiratory disease caused by the MERS coronavirus (MERS-CoV). MERS has been endemic to Saudi Arabia since 2012. The reservoir of MERS-CoV is the dromedary camel, suggesting that MERS is primarily a zoonotic disease. MERS-CoV is common in dromedaries throughout the Middle East, North Africa, and East Africa as evidenced by neutralizing antibodies against MERS-CoV; however, human cases have remained limited to the Middle East. To better understand the cause of this difference, the virological properties of African camel MERS-CoV were analyzed based on the spike (S) protein in Ethiopia. Nasal swabs were collected from 258 young dromedaries (≤ 2 years old) in the Afar region of Ethiopia, of which 39 were positive for MERS-CoV, as confirmed by genetic tests. All positive tests were exclusive to the Amibara woreda region. Using next-generation sequencing, two full-length genomes of Amibara isolates were successfully decoded; both isolates belonged to the C2 clade based on phylogenetic analysis of full-length and S protein sequences. Recombinant EMC isolates of MERS-CoV, in which the S protein is replaced with those of Amibara isolates, were then generated to test the roles of these proteins in viral properties. Amibara S recombinants replicated more slowly in cultured cells than in EMC S recombinants. In neutralizing assays, Amibara S recombinants were neutralized by lower concentrations of sera from both Ethiopian dromedaries and EMC isolate (wild-type)-immunized mouse sera, relative to the EMC S recombinants, indicating that viruses coated in the Amibara S protein were easier to neutralize than the EMC S protein. Neutralization experiments performed using S1/S2 chimeric recombinants of the EMC and Amibara S proteins showed that the neutralization profile was dependent on the S1 region of the S protein. These results suggest that the slower viral replication and the ease of neutralization seen in the Ethiopian MERS-CoV are due to strain-specific differences in the S protein and may account for the absence of human MERS-CoV cases in Ethiopia.

Transcriptional activation of long terminal repeat of bovine leukemia virus by bovine heat shock factor 1.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis (EBL). The BLV genome encodes Tax protein, a transcriptional activator of viral gene expression that binds to the BLV long terminal repeat (LTR). Heat shock factor 1 (HSF1) is a known regulator of the heat shock response proteins, including heat shock proteins. In the present study, the BLV LTR was investigated for interaction of heat shock element (HSE) with HSF1 and the viral Tax protein. It could be confirmed that a functional HSE is well conserved in different BLV strains. The LTR transcriptional activity, as measured by luciferase reporter assay, was upregulated by bovine HSF1 - without Tax expression - in feline CC81 cells. The HSF1 activated LTR transcription by binding to the HSE. LTR-activation was lost upon HSE removal from the LTR and upon expression of a mutant HSF1 lacking the DNA-binding domain. We conclude that BLV LTR is activated to a basal level by host transcriptional factor HSF1, but without Tax protein involvement.

A sensitive luminescence syncytium induction assay (LuSIA) based on a reporter plasmid containing a mutation in the glucocorticoid response element in the long terminal repeat U3 region of bovine leukemia virus.

BACKGROUND: Bovine leukemia virus (BLV) causes enzootic bovine leukosis, the most common neoplastic disease of cattle. Previously, we reported the luminescence syncytium induction assay (LuSIA), an assay for BLV infectivity based on CC81-BLU3G cells, which form syncytia expressing enhanced green fluorescent protein (EGFP) when co-cultured with BLV-infected cells. To develop a more sensitive LuSIA, we here focused on the glucocorticoid response element (GRE) within the U3 region of the BLV long terminal repeat (LTR). METHODS: We changed five nucleotide sites of the GRE in a pBLU3-EGFP reporter plasmid containing the BLV-LTR U3 region promoter by site-directed mutagenesis and we then constructed a new reporter plasmid (pBLU3GREM-EGFP) in which the EGFP reporter gene was expressed under control of the GRE-mutated LTR-U3 promoter. We also established a new CC81-derived reporter cell line harboring the GRE-mutated LTR-U3 promoter (CC81-GREMG). To evaluate the sensibility, the utility and the specificity of the LuSIA using CC81-GREMG, we co-cultured CC81-GREMG cells with BLV-persistently infected cells, free-viruses, white blood cells (WBCs) from BLV-infected cows, and bovine immunodeficiency-like virus (BIV)- and bovine foamy virus (BFV)-infected cells. RESULTS: We successfully constructed a new reporter plasmid harboring a mutation in the GRE and established a new reporter cell line, CC81-GREMG; this line was stably transfected with pBLU3GREM-EGFP in which the EGFP gene is expressed under control of the GRE-mutated LTR-U3 promoter and enabled direct visualization of BLV infectivity. The new LuSIA protocol using CC81-GREMG cells measures cell-to-cell infectivity and cell-free infectivity of BLV more sensitively than previous protocol using CC81-BLU3G. Furthermore, it did not respond to BIV and BFV infections, indicating that the LuSIA based on CC81-GREMG is specific for BLV infectivity. Moreover, we confirmed the utility of a new LuSIA based on CC81-GREMG cells using white blood cells (WBCs) from BLV-infected cows. Finally, the assay was useful for assessing the activity of neutralizing antibodies in plasma collected from BLV-infected cows. CONCLUSION: The new LuSIA protocol is quantitative and more sensitive than the previous assay based on CC81-BLU3G cells and should facilitate development of several new BLV assays.

Influenza D Virus Infection in Dromedary Camels, Ethiopia.

Influenza D virus has been found to cause respiratory diseases in livestock. We surveyed healthy dromedary camels in Ethiopia and found a high seroprevalence for this virus, in contrast to animals co-existing with the camels. Our observation implies that dromedary camels may play an important role in the circulation of influenza D virus.

Isolation of bovine foamy virus in Japan.

Bovine foamy virus (BFV) is endemic in many countries, but has not been reported in Japan. A syncytium-forming virus was isolated from peripheral blood leukocytes of clinically healthy cattle on a farm in Kanagawa prefecture during a periodic epidemiological survey of viral diseases. The isolate was propagated in primary fetal bovine muscle cells and subsequently passaged in Madin-Darby bovine kidney cells. Since the isolate appeared to be distinct from the viruses with syncytium-forming ability previously isolated in Japan, we attempted to identify it using genomic analyses and electron microscopy. A phylogenetic analysis revealed that the isolate belongs to the bovine foamy virus cluster and is highly similar to a BFV strain isolated in China. A sero-epidemiological survey was performed using agar gel immunodiffusion test with the isolated virus as the antigen, and five of the 57 cattle tested were found to be seropositive.

Variations in the viral genome and biological properties of bovine leukemia virus wild-type strains.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis (EBL), which causes enormous economic losses in the livestock industry worldwide. To reduce the economic loss caused by BLV infection, it is important to clarify the characters associated with BLV transmissibility and pathogenesis in cattle. In this study, we focused on viral characters and examined spontaneous mutations in the virus and viral properties by analyses of whole genome sequences and BLV molecular clones derived from cows with and without EBL. Genomic analysis indicated that all 28 strains harbored limited genetic variations but no deletion mutations that allowed classification into three groups (A, B, and C), except for one strain. Some nucleotide/amino acid substitutions were specific to a particular group. On the other hand, these genetic variations were not associated with the host bovine leukocyte antigen-DRB3 allele, which is known to be related to BLV pathogenesis. The viral replication activity in vitro was high, moderate, and low in groups A, B, and C, respectively. In addition, the proviral load, which is related to BLV transmissibility and pathogenesis, was high in cows infected with group A strains and low in those infected with group B/C strains. Therefore, these results suggest that limited genetic variations could affect viral properties relating to BLV transmissibility and pathogenesis.

Mutation of the S and 3c genes in genomes of feline coronaviruses.

Feline coronavirus (FCoV) is classified into two biotypes based on its pathogenicity in cats: a feline enteric coronavirus of low pathogenicity and a highly virulent feline infectious peritonitis virus. It has been suspected that FCoV alters its biotype via mutations in the viral genome. The S and 3c genes of FCoV have been considered the candidates for viral pathogenicity conversion. In the present study, FCoVs were analyzed for the frequency and location of mutations in the S and 3c genes from faecal samples of cats in an animal shelter and the faeces, effusions, and tissues of cats that were referred to veterinary hospitals. Our results indicated that approximately 95% FCoVs in faeces did not carry mutations in the two genes. However, 80% FCoVs in effusion samples exhibited mutations in the S and 3c genes with remainder displaying a mutation in the S or 3c gene. It was also suggested that mutational analysis of the 3c gene could be useful for studying the horizontal transmission of FCoVs in multi-cat environments.

Characterization of novel monoclonal antibodies against the MERS-coronavirus spike protein and their application in species-independent antibody detection by competitive ELISA.

Since discovering the Middle East respiratory syndrome coronavirus (MERS-CoV) as a causative agent of severe respiratory illness in the Middle East in 2012, serological testing has been conducted to assess antibody responses in patients and to investigate the zoonotic reservoir of the virus. Although the virus neutralization test is the gold standard assay for MERS diagnosis and for investigating the zoonotic reservoir, it uses live virus and so must be performed in high containment laboratories. Competitive ELISA (cELISA), in which a labeled monoclonal antibody (MAb) competes with test serum antibodies for target epitopes, may be a suitable alternative because it detects antibodies in a species-independent manner. In this study, novel MAbs against the spike protein of MERS-CoV were produced and characterized. One of these MAbs was used to develop a cELISA. The cELISA detected MERS-CoV-specific antibodies in sera from MERS-CoV-infected rats and rabbits immunized with the spike protein of MERS-CoV. The MAb-based cELISA was validated using sera from Ethiopian dromedary camels. Relative to the neutralization test, the cELISA detected MERS-CoV-specific antibodies in 66 Ethiopian dromedary camels with a sensitivity and specificity of 98% and 100%, respectively. The cELISA and neutralization test results correlated well (Pearson's correlation coefficients=0.71-0.76, depending on the cELISA serum dilution). This cELISA may be useful for MERS epidemiological investigations on MERS-CoV infection.

Serological evidence of hepatitis E virus infection in dromedary camels in Ethiopia.

The genome of dromedary camel hepatitis E virus (DcHEV) has been detected in stool and serum samples from dromedary camels, but the sero-epidemiological information of DcHEV infection remains unclear. A total of 246 serum samples collected from dromedary camels (Camelus dromedarius) in Ethiopia, and 40 serum samples from Bactrian camels (Camelus ferus) in Mongolia were examined for the detection of anti-DcHEV IgG antibody by a newly developed enzyme-linked immunosorbent assay (ELISA) by using DcHEV-like particles (DcHEV-LPs) as the antigen. The results revealed that 55 of the 246 (22.4%) dromedary camels were positive for anti-DcHEV IgG, whereas all 40 samples from the Bactrian camels were negative for DcHEV IgG antibody. A total of 98 serum samples from dromedary camels, including 25 anti-DcHEV-IgG positive samples, were used for the detection of DcHEV RNA by reverse transcription-polymerase chain reaction (RT-PCR), however, no positive samples were identified. These results suggested that the DcHEV infection occurred in the dromedary camels in Ethiopia. Further studies are required to determine whether Bactrian camels are susceptible to DcHEV infection. In addition, not only DcHEV-LPs, but also virus-like particles (VLPs) delivered from G1, G3 and G5 HEV are likely applicable for the detection of the anti-DcHEV IgG antibody.

Enzootic bovine leukosis in a two-month-old calf.

A two-month-old calf was diagnosed with leukosis on the basis of the clinical sign of enlarged, superficial lymph nodes. Serological and genetic tests for bovine leukemia virus (BLV) were performed because the calf was born from a cow infected with BLV. The serum had a weakly positive BLV antibody, and the BLV provirus was detected within neoplastic cells on performing polymerase chain reaction (PCR). Analysis of the BLV provirus integration site using inverse PCR revealed that the BLV integration site location was identical on all chromosomes in all tumor tissues examined. Thus, the tumor cells monoclonally proliferated following BLV infection. The present study shows that enzootic bovine leukosis can occur in a young animal, as in the two-month-old calf in our study.

Preparation of a cell line persistently infected with maedi/visna virus and production of viral antigens.

We attempted to prepare a cell line that produces maedi/visna virus (MVV) and is free of contamination by other viruses and mycoplasmas. Three cell lines, which originated from a sheep, goat and bat, were infected with MVV and passaged approximately every 5 days. The cultured cells were then subjected to polymerase chain reaction analysis for MVV provirus. As a result, a cell line persistently infected with MVV was established from ZZ-R cells, which originated from the fetal goat tongue. The 50-fold concentrated culture fluid formed a precipitation line against reference antiserum.

Serological survey of viral diseases relating to reproductive failure among Artiodactyla in Ethiopian Camelus dromedarius.

Serological surveys were performed on Ethiopian camels with a history of abortion to investigate the presence of antibodies against viruses that infect animals classified in the order Artiodactyla. In 2013, 120 serum samples were collected from camels in various parts of Ethiopia. Several viruses related to abortion in ruminants were prevalent. In particular, antibodies against bluetongue virus, were detected at a high rate (76.7% of samples). Additionally, antibodies against Akabane virus and Japanese encephalitis virus were also detected in samples from more than 40% of the camels; however, their antibody titers were relatively low.

Survey on vertical infection of bovine viral diarrhea virus from fetal bovine sera in the field.

Bovine viral diarrhea virus (BVDV) isolation and antibody survey were performed using 2,758 fetal bovine sera (FBS) collected from slaughterhouses in New Zealand, Australia and the Dominican Republic, and then sent to Japan to manufacture commercial serum for cell culture use. FBS in the Dominican Republic were pooled for each several individuals, and those collected in other countries were separated according to each individual and subjected to the tests. BVDV was isolated from 25 (0.91%) FBS, and the BVDV antibody was detected in 44 (1.60%) FBS. The survey on 139 sets of paired sera of a dam and her fetus revealed that neither the BVDV antibody nor BVDV was detected in all FBS from BVDV antibody-positive dams.

Overexpression of feline tripartite motif-containing 25 interferes with the late stage of feline leukemia virus replication.

Tripartite motif-containing 25 (TRIM25) regulates various cellular processes through E3 ubiquitin ligase activity. Previous studies have revealed that the expression of TRIM25 is induced by type I interferon and that TRIM25 is involved in the host cellular innate immune response against retroviral infection. Although retroviral infection is prevalent in domestic cats, the roles of feline TRIM25 in the immune response against these viral infections are poorly understood. Because feline TRIM25 is expected to modulate the infection of feline leukemia virus (FeLV), we investigated its effects on early- and late-stage FeLV replication. This study revealed that ectopic expression of feline TRIM25 in HEK293T cells reduced viral protein levels leading to the inhibition of FeLV release. Our findings show that feline TRIM25 has a potent antiviral activity and implicate an antiviral mechanism whereby feline TRIM25 interferes with late-stage FeLV replication.

Isolation of maedi/visna virus from a sheep in Japan.

Maedi/visna (MV) is a lentiviral disease of sheep caused by the maedi/visna virus (MVV). Although MV is prevalent in many countries, it had not been reported in Japan. In 2011, however, three sheep in northern Japan were reported to be seropositive against the MVV antigen, indicating a persistent MVV infection. In the present study, we isolated MVV from one sheep to confirm MVV infection and conducted genomic classification of the virus. The co-culture of leukocytes from a seropositive sheep with fetal goat lung cells resulted in the formation of syncytial cells and the amplification of a long terminal repeat sequence of MVV by polymerase chain reaction. The isolate was confirmed as being MVV, rather than the caprine arthritis-encephalitis virus based on phylogenetic analysis of the gag gene sequence. Although the sheep was asymptomatic, nonpurulent meningitis and demyelination were found in the spinal cord. These were considered to be early lesions associated with pathogenic MVV infection. Therefore, the present study demonstrated that MVV is distributed in Japan.

Efficient propagation of equine viruses in a newly established equine cell line, FHK-13.1 cells.

Equine cells are required for isolation of viruses that infect the horse. However, only a few equine cell lines and cell cultures are available so far. Fetal horse kidney (FHK)-Tcl3.1 cell is a novel cell line established by introducing simian virus 40 (SV40) large T antigen. In the present study, the ability to propagate equine viruses was compared between FHK-Tcl3.1 cells and other equine cells. FHK-Tcl3.1 cells efficiently increased many viruses derived from or having pathogenicity to horses and produced high infective titers in culture fluids. These results indicate that FHK-Tcl3.1 cells would be useful for propagation and serological tests of viruses that affect Equidae.