Antiviral activity of bovine type III interferon against bovine viral diarrhea virus is greatly reduced in bovine turbinate cells due to limited expression of IFN lambda receptor 1 (IL-28Rα).

Introduction: The antiviral activity of recombinant bovine interferon lambda 3 (bovIFN-λ3) against bovine viral diarrhea virus (BVDV) has been demonstrated in vitro in Madin-Darby bovine kidney cells (MDBK) and in vivo in cattle. However, anti-BVDV activity of bovIFN-λ3 has not been studied in bovine respiratory tract epithelial cells, supposedly a primary target of BVDV infection when entering the host by the oronasal route. Methods: Here we investigated the anti-BVDV activity of bovIFN-λ3 in bovine turbinate-derived primary epithelial cells (BTu) using BVDV infection and immunoperoxidase staining, TCID50, RT-qPCR, DNA and transcriptome sequencing, and transfection with plasmids containing the two subunits, IL-28Rα and IL-10Rß that constitute the bovIFN-λ3 receptor. Results: Our immunoperoxidase staining, RT-qPCR, and TCID50 results show that while BVDV was successfully cleared in MDBK cells treated with bovIFN-λ3 and bovIFN-α, only the latter, bovIFN-α, cleared BVDV in BTu cells. Preincubation of MDBK cells with bovIFN-λ3 before BVDV infection was needed to induce optimal antiviral state. Both cell types displayed intact type I and III IFN signaling pathways and expressed similar levels of IL-10Rß subunit of the type III IFN receptor. Sequencing of PCR amplicon of the IL-28Rα subunit revealed intact transmembrane domain and lack of single nucleotide polymorphisms (SNPs) in BTu cells. However, RT-qPCR and transcriptomic analyses showed a lower expression of IL-28Rα transcripts in BTu cells as compared to MDBK cells. Interestingly, transfection of BTu cells with a plasmid encoding IL-28Rα subunit, but not IL-10Rß subunit, established the bovIFN-λ3 sensitivity showing similar anti-BVDV activity to the response in MDBK cells. Conclusion: Our results demonstrate that the sensitivity of cells to bovIFN-λ3 depends not only on the quality but also of the quantity of the IL-28Rα subunit of the heterodimeric receptor. A reduction in IL-28Rα transcript expression was detected in BTu as compared to MDBK cells, despite the absence of spliced variants or SNPs. The establishment of bovIFN-λ3 induced anti-BVDV activity in BTu cells transfected with an IL-28Rα plasmid suggests that the level of expression of this receptor subunit is crucial for the specific antiviral activity of type III IFN in these cells.

Positively Charged Amino Acids in the Pestiviral Erns Control Cell Entry, Endoribonuclease Activity and Innate Immune Evasion.

The genus Pestivirus, family Flaviviridae, includes four economically important viruses of livestock, i.e., bovine viral diarrhea virus-1 (BVDV-1) and -2 (BVDV-2), border disease virus (BDV) and classical swine fever virus (CSFV). Erns and Npro, both expressed uniquely by pestiviruses, counteract the host's innate immune defense by interfering with the induction of interferon (IFN) synthesis. The structural envelope protein Erns also exists in a soluble form and, by its endoribonuclease activity, degrades immunostimulatory RNA prior to their activation of pattern recognition receptors. Here, we show that at least three out of four positively-charged residues in the C-terminal glycosaminoglycan (GAG)-binding site of BVDV-Erns are required for efficient cell entry, and that a positively charged region more upstream is not involved in cell entry but rather in RNA-binding. Moreover, the C-terminal domain on its own determines intracellular targeting, as GFP fused to the C-terminal amino acids of Erns was found at the same compartments as wt Erns. In summary, RNase activity and uptake into cells are both required for Erns to act as an IFN antagonist, and the C-terminal amphipathic helix containing the GAG-binding site determines the efficiency of cell entry and its intracellular localization.

Hyper-Enriched Anti-RSV Immunoglobulins Nasally Administered: A Promising Approach for Respiratory Syncytial Virus Prophylaxis.

Respiratory syncytial virus (RSV) is a public health concern that causes acute lower respiratory tract infection. So far, no vaccine candidate under development has reached the market and the only licensed product to prevent RSV infection in at-risk infants and young children is a monoclonal antibody (Synagis®). Polyclonal human anti-RSV hyper-immune immunoglobulins (Igs) have also been used but were superseded by Synagis® owing to their low titer and large infused volume. Here we report a new drug class of immunoglobulins, derived from human non hyper-immune plasma that was generated by an innovative bioprocess, called Ig cracking, combining expertises in plasma-derived products and affinity chromatography. By using the RSV fusion protein (F protein) as ligand, the Ig cracking process provided a purified and concentrated product, designated hyper-enriched anti-RSV IgG, composed of at least 15-20% target-specific-antibodies from normal plasma. These anti-RSV Ig displayed a strong in vitro neutralization effect on RSV replication. Moreover, we described a novel prophylactic strategy based on local nasal administration of this unique hyper-enriched anti-RSV IgG solution using a mouse model of infection with bioluminescent RSV. Our results demonstrated that very low doses of hyper-enriched anti-RSV IgG can be administered locally to ensure rapid and efficient inhibition of virus infection. Thus, the general hyper-enriched Ig concept appeared a promising approach and might provide solutions to prevent and treat other infectious diseases. IMPORTANCE: Respiratory Syncytial Virus (RSV) is the major cause of acute lower respiratory infections in children, and is also recognized as a cause of morbidity in the elderly. There are still no vaccines and no efficient antiviral therapy against this virus. Here, we described an approach of passive immunization with a new class of hyper-enriched anti-RSV immunoglobulins (Ig) manufactured from human normal plasma. This new class of immunoglobulin plasma derived product is generated by an innovative bioprocess, called Ig cracking, which requires a combination of expertise in both plasma derived products and affinity chromatography. The strong efficacy in a small volume of these hyper-enriched anti-RSV IgG to inhibit the viral infection was demonstrated using a mouse model. This new class of immunoglobulin plasma-derived products could be applied to other pathogens to address specific therapeutic needs in the field of infectious diseases or even pandemics, such as COVID-19.

Isolation and development of bovine primary respiratory cells as model to study influenza D virus infection.

Influenza D virus (IDV) is a novel type of influenza virus that infects and causes respiratory illness in bovines. Lack of host-specific in vitro model that can recapitulate morphology and physiology of in vivo airway epithelial cells has impeded the study of IDV infection. Here, we established and characterized bovine primary respiratory epithelial cells from nasal turbinate, soft palate, and trachea of the same calf. All three cell types showed characteristics peculiar of epithelial cells, polarized into apical-basolateral membrane, and formed tight junctions. Furthermore, these cells expressed both α-2,3- and α-2,6-linked sialic acids with α-2,3 linkage being more abundant. IDV strains replicated to high titers in these cells, while influenza A and B viruses exhibited moderate to low titers, with influenza C virus replication not detected. These findings suggest that bovine primary airway epithelial cells can be utilized to model infection biology and pathophysiology of IDV and other respiratory pathogens.

Expression of Sp100 Protein in Human Papillomavirus-Associated Sinonasal Inverted Papilloma.

OBJECTIVE: Sinonasal inverted papilloma (SNIP) is a benign tumor characterized by an aggressive growth, a tendency to recur, and an association with malignancy. However, the precise etiology of SNIP is still unknown. The objective of this study was to identify the expression pattern of speckled protein 100 (Sp100) in the malignant transformation (MT) of SNIP and its correlation with human papillomavirus (HPV)-16 and HPV-18 infections and other clinical features. This would further help in understanding the possible mechanisms for the development of SNIP. METHODS: Individual nasal mucosa specimens from 40 patients (25 males and 15 females) and 10 inferior turbinate specimens as controls were included in the present study. The samples were divided into several sections for histopathological examination, HPV DNA detection, and immunohistochemical staining. RESULTS: We observed that as SNIP progressed, the Sp100 protein expression was gradually downregulated, and SP100 localization changed from nucleus to the cytoplasm. Positive rate of HPV infection in the SNIP with MT group was higher than that in the other groups, and Sp100 expression was correlated to HPV infections and SNIP with MT. However, no correlation was observed between Sp100 expression and clinical features, such as age, gender, and smoking. CONCLUSION: Positive rate of HPV infection is high in the SNIP with MT and has a correlation with Sp100 expression. In addition, the expression of Sp100 is downregulated in SNIP with MT, and Sp100 may play a role in the progression of SNIP.

Immune efficacy of an adenoviral vector-based swine influenza vaccine against antigenically distinct H1N1 strains in mice.

Avian-like H1N1 swine influenza viruses are prevalent in pigs and have occasionally crossed the species barrier and infected humans, which highlights the importance of preventing swine influenza. Human adenovirus serotype 5 (Ad5) has been tested in human influenza vaccine clinical trials and has exhibited a reliable safety profile. Here, we generated a replication-defective, recombinant adenovirus (designated as rAd5-avH1HA) expressing the hemagglutinin gene of an avian-like H1N1 virus (A/swine/Zhejiang/199/2013, ZJ/199/13). Using a BALB/c mouse model, we showed that a two-dose intramuscular administration of recombinant rAd5-avH1HA induced high levels of hemagglutination inhibition antibodies and prevented homologous and heterologous H1N1 virus-induced weight loss, as well as viral replication in the nasal turbinates and lungs of mice. Furthermore, a prime-boost immunization strategy trial with a recombinant plasmid (designated as pCAGGS-HA) followed by rAd5-avH1HA vaccine provided effective protection against homologous and heterologous H1N1 virus infection in mice. These results indicate that rAd5-avH1HA is an efficacious genetically engineered vaccine candidate against H1N1 swine influenza. Future studies should examine its immune efficacy in pigs.

1,8-Cineole potentiates IRF3-mediated antiviral response in human stem cells and in an ex vivo model of rhinosinusitis.

The common cold is one of the most frequent human inflammatory diseases caused by viruses and can facilitate bacterial superinfections, resulting in sinusitis or pneumonia. The active ingredient of the drug Soledum, 1,8-cineole, is commonly applied for treating inflammatory diseases of the respiratory tract. However, the potential for 1,8-cineole to treat primary viral infections of the respiratory tract remains unclear. In the present study, we demonstrate for the first time that 1,8-cineole potentiates poly(I:C)-induced activity of the antiviral transcription factor interferon regulatory factor 3 (IRF3), while simultaneously reducing proinflammatory nuclear factor (NF)-κB activity in human cell lines, inferior turbinate stem cells (ITSCs) and in ex vivo cultivated human nasal mucosa. Co-treatment of cell lines with poly(I:C) and 1,8-cineole resulted in significantly increased IRF3 reporter gene activity compared with poly(I:C) alone, whereas NF-κB activity was reduced. Accordingly, 1,8-cineole- and poly(I:C) treatment led to increased nuclear translocation of IRF3 in ITSCs and a human ex vivo model of rhinosinusitis compared with the poly(I:C) treatment approach. Nuclear translocation of IRF3 was significantly increased in ITSCs and slice cultures treated with lipopolysaccharide (LPS) and 1,8-cineole compared with the LPS-treated cells mimicking bacterial infection. Our findings strongly suggest that 1,8-cineole potentiates the antiviral activity of IRF3 in addition to its inhibitory effect on proinflammatory NF-κB signalling, and may thus broaden its field of application.

Comparison of 'HoBi'-like viral populations among persistent infected calves generated under experimental conditions and to inoculum virus.

Like other members from the Pestivirus genus, 'HoBi'-like pestiviruses cause economic losses for cattle producers due to both acute and persistent infections. The present study analyzed for the first time PI animals derived from a controlled infection with two different 'HoBi'-like strains where the animals were maintained under conditions where superinfection by other pestiviruses could be excluded. The sequence of the region coding for viral glycoproteins E1/E2 of variants within the swarms of viruses present in the PI calves and two viral inoculums used to generate them were compared. Differences in genetic composition of the viral swarms were observed suggesting that host factors can play a role in genetic variations among PIs. Moreover, PIs generated with the same inoculum showed amino acid substitutions in similar sites of the polyprotein, even in serum from PIs with different quasispecies composition, reinforcing that some specific sites in E2 are important for host adaptation.

Prevalence of human papillomavirus, Epstein-Barr virus, p21, and p53 expression in sinonasal inverted papilloma, nasal polyp, and hypertrophied turbinate in Hong Kong patients.

BACKGROUND: The purpose of this study of human papillomavirus (HPV), Epstein-Barr virus (EBV), p21, and p53 in sinonasal inverted papilloma (IP) was to help elucidate its pathogenesis. METHODS: Seventy-three IPs, 48 nasal polyps, and 85 hypertrophied turbinates were subjected to HPV polymerase chain reaction (PCR) study. Seventy-three IPs, 30 nasal polyps, and 32 hypertrophied turbinates were subjected to EBV in situ hybridization (ISH), p21, and p53 immunohistochemical (IHC) studies. RESULTS: HPV was positive in 3 of 73 IPs (4.1%). All specimens were EBV negative. In all, 99% of IPs showed strong and diffuse p21 nuclear reactivity. Most nasal polyps and hypertrophied turbinates showed weak to moderate immunoreactivity of the basal and parabasal cells. Only focal p53 immunoreactivity of the basal and parabasal cells was found in 19% of IPs and 40% of nasal polyps. CONCLUSIONS: HPV prevalence of our IP is low. EBV is not present in IP. High p21 and low p53 expression in IP suggests a non-p53-dependent regulation pathway.

Prevalence of human papilloma virus and human herpes virus types 1-7 in human nasal polyposis.

This study aimed to investigate the prevalence of human papilloma virus (HPV), herpes simplex virus-1/-2 (HSV-1/-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpes virus-6/-7 (HHV-6/-7) in 23 human nasal polyps by applying PCR. Two types of control tissues were used: adjacent inferior/middle turbinates from the patients and inferior/middle turbinates from 13 patients undergoing nasal corrective surgery. EBV was the virus most frequently detected (35%), followed by HPV (13%), HSV-1 (9%), and CMV (4%). The CMV-positive polyp was simultaneously positive for HSV-1. HPV was also detected in the adjacent turbinates (4%) and the adjacent middle turbinate (4%) of one of the HPV-positive patients. EBV, HSV, and CMV were not detected in the adjacent turbinates of the EBV-, HSV- or CMV-positive patients. All mucosae were negative for the VZV, HHV-6, and HHV-7. This is the first study to deal with the involvement of a comparable group of viruses in human nasal polyposis. The findings support the theory that the presence of viral EBV markedly influences the pathogenesis of these benign nasal tumors. The low incidence of HPV detected confirms the hypothesis that HPV is correlated with infectious mucosal lesions to a lesser extent than it is with proliferative lesions, such as inverted papilloma. The low incidence of HSV-1 and CMV confirms that these two herpes viruses may play a minor role in the development of nasal polyposis. Double infection with HSV-1 and CMV may also play a minor, though causative, role in nasal polyp development. VZV and HHV-6/-7 do not appear to be involved in the pathogenesis of these mucosal lesions.

Detection of ovine herpesvirus 2 major capsid gene transcripts as an indicator of virus replication in shedding sheep and clinically affected animals.

The aim of this study was to identify tissues where ovine herpesvirus 2 (OvHV-2) replication occurs in vivo. A reverse-transcriptase PCR targeting the OvHV-2 major capsid protein gene (ORF 25) was developed and the presence of transcripts used as an indicator of virus replication in naturally infected sheep, and cattle and bison with sheep-associated malignant catarrhal fever (SA-MCF). ORF 25 transcripts were detected in 18 of 60 (30%) turbinate, trachea, and lung samples from five sheep experiencing a shedding episode; 12 of the 18 positive samples were turbinates. ORF 25 transcripts were not detected in any other tissue from the shedding sheep (n=55). In contrast, 86 of 102 (84%) samples from clinically affected bovine and bison tissues, including brain, kidney, intestine, and bladder, had ORF 25 transcripts. The data strongly suggest that OvHV-2 replication is localized to the respiratory tract of shedding sheep, predominantly in the turbinate, while it occurs in virtually all tissues of cattle and bison with SA-MCF. These findings represent an important initial step in understanding viral pathogenesis, and in potentially establishing a system for OvHV-2 propagation in vitro.

Detection of rhinovirus in turbinate epithelial cells of chronic sinusitis.

BACKGROUND: In contrast to the well-established association of rhinovirus (RV) with acute sinusitis, little is known about the role of RV infections in the pathogenesis of chronic sinusitis. Therefore, we assayed the nasal cavity mucosae of chronic sinusitis patients lacking signs of acute viral infection for the presence of RV. METHODS: Nasal lavage fluids and turbinate epithelial cells from 39 sinusitis patients and 27 control subjects were tested. Turbinate epithelial cells were collected using a Rhino-probe mucosal curette. Picornavirus was assayed by an initial reverse-transcription polymerase chain reaction (PCR), and picornavirus-positive samples were assayed by nested reverse-transcription PCR to detect RV. RESULTS: All lavage fluids from both groups, as well as control epithelial cells, were negative for picornavirus. In contrast, 8 of 39 (21%) epithelial cell samples from sinusitis patients were positive for picornavirus. RV-specific nested-PCR revealed that all eight of these samples were positive for RV. CONCLUSION: The detection of RV in the turbinate epithelial cells of chronic sinusitis patients suggests that RV may be important in the pathogenesis of chronic sinusitis.

Development of rhinovirus study model using organ culture of turbinate mucosa.

To better understand the pathophysiology of rhinovirus (RV) infection, a development of a study model using organ culture of turbinate mucosa was sought. Inferior turbinate mucosal tissues were cultured using air-liquid interface methods, on a support of gelfoam soaked in culture media. RV-16 was applied to the mucosal surface and washed off, and histological changes were evaluated. The success of RV infection was assayed by semi-nested RT-PCR of the mucosal surface fluid taken 48h after incubation. Intracellular RVs were visualized by in situ hybridization (ISH). Secretion of the cytokines, IL-6 and IL-8, into the culture media was quantitated by ELISA. After 7 days of culture, the turbinate mucosae did not show significant damage. A PCR product indicating successful RV infection was detected in 5 out of 10 mucosal tissues. ISH showed a very small number of positively stained cells focally located in the epithelial layer. In the beginning 24h after infection, secretion of IL-6 and IL-8 into the culture media of infected mucosae was significantly greater than into the media of control mucosae. Our results indicate that the air-liquid interface organ culture of turbinate mucosa could serve as an acceptable in vitro model for studying RV infection.

An evaluation of the genetic stability and pathogenicity of the Russian cold-adapted influenza A donor strains A/Leningrad/134/17/57 and A/Leningrad/134/47/57 in ferrets.

The influenza A components of live attenuated vaccines used in Russia have been prepared as reassortants of the cold-adapted (ca) H2N2 viruses, A/Leningrad/134/17/57-ca (Len/17) and A/Leningrad/134/47/57-ca (Len/47), and virulent epidemic strains. The lesions responsible for attenuation within the six internal genes of each donor strain have been sequenced and described, but relatively little is known as to their stability before and after passage in susceptible hosts. In the work reported in this paper, RT-PCR restriction analysis and limited sequencing of individual genes were used to evaluate the stability of lesions in stocks of the both donor strains after passage in ferrets, which have been used widely as susceptible hosts for assessment of the virulence of influenza strains. Len/47 was shown to possess expected lesions by RT-PCR and restriction analysis. Substitution at position 1066 of the NP gene, which has been previously reported to be unique to Len/47 [Klimov et al., Virology 186 (1992) 795], was also shown to be present in all clones of Len/17. This change was confirmed by limited sequence analysis and was shown to be retained in progeny viruses isolated from the lungs and turbinates of inoculated ferrets. Two other changes in the PB2 and PB1 genes that were present in Len/47 were detected by limited sequence analysis alone. Further previously unreported minor changes were shown to be present for Len/17 and Len/47, but not both, and their significance is unknown. Limited replication of each donor strain occurred in ferrets and minimal clinical signs and histopathology were present. By contrast, the parental strain Len/57 and the recent epidemic strain A/Sydney/6/97 induced clinical signs and histopathology that were typical of influenza disease.

Genetic analysis of the G and P genes in ungulate respiratory syncytial viruses by RNase A mismatch cleavage method.

The G and P genes of bovine, ovine and caprine respiratory syncytial (RS) viruses were analyzed by RNase A one-dimensional fingerprinting, using A 51908 as the reference strain. Antisense G or P RNA probes of bovine RS virus strain A 51908 were hybridized to total RNA extracted from bovine turbinate cells infected with bovine, ovine or caprine RS virus strains. The RNA:RNA heteroduplexes were digested with RNase A and the resistant products were analyzed by gel electrophoresis. Comparative analysis of the cleavage patterns revealed heterogeneity among bovine, ovine and caprine RS virus isolates. Ovine RS virus strains generated RNA cleavage patterns more distantly related to the bovine or caprine RS virus strains, particularly in the G gene. Statistical analysis of the results obtained indicated that genetic differences between bovine and ovine viruses were larger, compared with the ones among bovine strains themselves. The same analysis also revealed a close genetic relation among bovine and caprine strains. These results are discussed in terms of ungulate RS virus genetic variation and vaccine development.

High level expression of equine herpesvirus 1 glycoproteins D and H and their role in protection against virus challenge in the C3H (H-2Kk) murine model.

N and C-terminal truncated forms of equine herpesvirus 1 (EHV 1) glycoproteins gD and gH were expressed in baculovirus resulting in the production of secreted recombinant proteins. A carboxy-terminal histidine tag was included on each of the genes for protein isolation by nickel affinity chromatography. Recombinant gD was recognized by three gD specific monoclonal antibodies, 20C4, 5H6 and F3132. F3132 is a conformationally dependent monoclonal antibody with virus neutralizing activity. Expression of gH was confirmed by reacting the protein with the gH peptide specific antiserum R319. The truncated gD gene was also expressed as a beta-galactosidase fusion protein which was purified from E. coli by nickel affinity chromatography. C3H mice were inoculated with purified recombinant gD or gH or insect cells which had been infected with recombinant baculoviruses. Mice were subsequently challenged with EHV 1. Purified recombinant baculovirus gD provided the most protection and produced high levels of virus neutralizing antibodies. The gD fusion protein was less effective at protecting mice and insect cells infected with either of the recombinant baculoviruses or purified recombinant gH were poor at conferring protection. The results emphasize the importance of using purified proteins in vaccine formulations and of including EHV 1 gD as a component of a subunit vaccine.

Detection of turkey rhinotracheitis virus from chickens with swollen head syndrome by reverse transcriptase-polymerase chain reaction (RT-PCR).

We developed a sensitive and specific method, reverse transcriptase-polymerase chain reaction (RT-PCR) method, for detection of turkey rhinotracheitis virus (TRTV). Two sets of primers were designed from F protein gene sequence of TRTV 3B strain. Sensitivity of detection by nested PCR with the primers corresponded to 0.4 TCID50. Applying this method to a field case of swollen head syndrome (SHS), TRTV could be detected directly from chicken trachea and turbinates. It was identified that this method was very useful to examine the relation of TRTV and SHS.

Localization of human rhinovirus replication in the upper respiratory tract by in situ hybridization.

To localize the sites and determine the extent of human rhinovirus (HRV) replication in the upper respiratory tract, biopsies of nasal and nasopharyngeal epithelia were collected from 26 HRV- or 7 sham-inoculated volunteers on days 1, 3, and 5 and on days 12, 20, or 33 after inoculation and analyzed by in situ hybridization. HRV-infected cells were detected on at least 1 day in 22 of the 23 HRV-infected subjects and in 1 of the 7 sham-inoculated subjects who developed a cold and had nasal secretions positive for a picornavirus by polymerase chain reaction. Low numbers of in situ hybridization-positive ciliated cells were present in nasal biopsies. In the nasopharynx, most HRV-infected cells were ciliated, but infected nonciliated epithelial cells were also detected. Our results indicate that HRV replicates in a very small proportion of cells in the nasal epithelium and in both ciliated and nonciliated cells in the nasopharynx of experimentally infected humans.