Influenzavirus A

An influenza-seronegative 10-week-old Beagle dog was experimentally inoculated with the avian origin canine influenza virus A/canine/01/2007, subtype H3N2, which was originally isolated from a pet dog with severe respiratory syndrome [2 (link)]. Eight hours after inoculation, the infected dog was cohoused with seven uninfected Beagle dogs in the animal facilities at Green Cross Veterinary Products (Yongin, South Korea). All animal experiments complied with the current laws of South Korea. Animal care and treatment were conducted in accordance with guidelines established by the Green Cross Veterinary Products Institutional Animal Care and Use Committee.
Clinical signs (sneezing, nasal discharge and coughing) and rectal temperatures were recorded daily for 14 days post-inoculation (DPI), and nasal swab samples for the detection of viral shedding were also collected daily over the same time course. Serum samples were collected on days 0, 7, and 14, and antibodies against nucleoprotein (NP) were detected by a commercial canine influenza virus (CIV) competitive ELISA (Animal Genetics, Inc., Suwon, South Korea) [1 (link)].
To investigate the correlation between fever and viral shedding, the mean viral titers in the nasal discharge were examined during fever (body temperature >39.5°C) and non-fever stages.

PB1, PB2, PA and NP-expressing plasmids of influenza virus A/WSN/33 (H1N1) pcDNA-PB1, pcDNA-PB2, pcDNA-PA, pcDNA NP, pcDNA-PB2-TAP and pcDNA-PA-TAP) have been described [17] (link), [47] (link), [58] (link). Full-length A/NT/60/68 (H3N2) PB1, PB2 and PA sequences were PCR amplified from the pBR322 clones, A/NT/60/68/2/62, A/NT/60/68/1 and A/NT/60/68/3/11 [accession numbers: J02138 (PB1), J02139 (PA) and J02140 (PB2)] [59] (link)–[61] (link) and inserted into pcDNA3A [45] (link) using KpnI and NotI restriction sites generating pcDNA/60/PB1, pcDNA/60/PA, pcDNA/60/PB2, pcDNA/60/PA-TAP and pcDNA/60/PB2-TAP expression vectors. To construct expression vectors for A/duck/Fujian/01/02 PB1, PB2 and PA and NP [accession numbers: AY585483 (PB1), AY585504 (PB2) and AY5854625 (PA) and AY585420 (NP)] [62] (link), full-length sequences were PCR amplified from pBD clones [63] (link). PB1, PA and NP PCR products were inserted into pcDNA3A using KpnI and NotI restriction sites generating pcDNA-FJ/01/02-PB1, pcDNA-FJ/01/02-PA and pcDNA-FJ/01/02-NP. PB2 was inserted into pcDNA3A and pcDNA-PB2-TAP using HindIII and NotI restriction sites generating pcDNA-FJ/01/02-PB2 and pcDNA-FJ/01/02-PB2-TAP, respectively.
To construct PB1, PB2 and PA expression vectors of A/HongKong/156/97(H5N1) [accession numbers: AF036362 (PB1), AF046095 (PA) and AF046093 (PB2)] [32] (link), [49] (link) and A/Vietnam/1194/04(H5N1) [accession numbers: AY651664 (PB1), AY651610 (PA) and AY651718 (PB2)] [64] (link), RT-PCR was performed with Superscript II reverse transcriptase (Invitrogen) and Fusion polymerase (Stratagene) with RNA isolated from virus grown in embryonated chicken eggs. PCR fragments were inserted into pCR2.1-TOPO (Invitrogen) by TA cloning, and the coding region subcloned into pcDNA3A using KpnI and NotI sites, generating pcDNA/156/PB1, pcDNA/156/PA, pcDNA/156/PB2, pcDNA/156/PA-TAP, pcDNA/156/PB2-TAP, pcDNA/1194/PB1, pcDNA/1194/PA, pcDNA/1194/PB2, pcDNA/1194/PA-TAP and pcDNA/1194/PB2-TAP.
The A/Hong Kong/156/97 PB1 cDNA cloned here has 4 mutations at the following nucleotide positions (counting from the A of the initiator ATG as nucleotide 1) compared to the sequence of AF036362 (i) 318, G→A (ii) 355, A→G (iii) 1828, G→T (iv) 2276, A→G, causing 2 coding changes at amino acid residues 119, Met→Val and 610, Gly→Cys. PB2 and PA nucleotide and amino acid sequences of A/Hong Kong/156/97 were identical to the databases AF046093 (PB2) and AF046095 (PA). A/Vietnam/1194/04 PB2 cDNA cloned here also two coding changes (H60D and K189E) compared to the sequence of AY651718. The pPOLI-vNA plasmid has been described previously [17] (link). The PRC425.vNA plasmid was created by subcloning the EcoRI and BpuAI fragment of the A/WSN/33 NA gene of pPOLI NA into pPRC425 [48] (link). The plasmid contains part of the sequence of the NA gene under the control of a chicken Pol I promoter. To construct PA chimera plasmids, the N-terminal half or the C-terminal half of WSN PA was amplified by PCR and inserted into pcDNA/156/PA at KpnI and BamHI sites, or BamHI and NotI sites, generating pcDNA/WH/PA and pcDNA/HW/PA, respectively. Point mutations in the WSN PA gene were made by site directed mutagenesis [6] (link), [17] (link), [23] (link) and were confirmed by full sequencing of the gene. Primer sequences are available upon request.

Across all sets (feature selection, training, extra-familial validation), six families of respiratory viruses were included in this study: Coronaviridae, Paramyxoviridae, Pneumoviridae, Adenoviridae, Orthomyxoviridae, and Herpesviridae. Each of the viruses within these families has a protein responsible for viral attachment and host cell entry, which will be referred to herein as the “spike” protein (see Fig 1A). For Coronaviruses, it is the Spike S Glycoprotein which is aptly named because it projects from the surface of the virion (Fig 1B) as do the other “spike” proteins. Note that for Influenza Virus A within the Orthomyxoviridae family, we selected Hemagglutinin as the equivalent of the “spike” over Neuraminidase as the latter primarily prevents virion aggregation and as such serves more as a helper protein to the role of the former in determining cell entry [25 (link)].
A total of 50 viral sequences (ranging from 4 to 12 for each virus family) encoding 360 proteins were utilized (see Table 1 for a list of sequences). Specifically, in the feature selection set we included 7 Coronaviridae sequences representing 7 viruses; in the training set, we included 7 different Coronaviridae sequences representing 7 viruses, 4 Paramyxoviridae sequences representing 4 viruses, 12 Pneumoviridae sequences representing 2 viruses, 8 Adenoviridae sequences representing 1 virus, and 8 Orthomyxoviridae sequences representing 1 virus. Finally, for the extra-familial validation set, we included 4 Herpesviridae sequences representing 4 viruses. See Table 2 for the number of “spike” vs. non-spike proteins for each virus family.

Sf9 insect cells were maintained in SF900II (Life Technologies, San Diego, CA, USA) at 27 °C in cell culture. Influenza virus A/Aichi/68 H3N2 and A/Anhui/2013 H7N9 provided by Dr. Zhu Hongwei (Ludong University) were propagated in the allantoic cavities of 10-day-old embryonated hen’s eggs at 37 °C for 2 d. Allantoic fluid was harvested and centrifuged at 2000 rpm for 10 min. The viruses were titrated by infection of mice with serial dilutions, and LD₅₀ (50% lethal dose) was calculated using the method of Reed and Muench.

To determine A/California/07/2009 NA activity, high-binding flat-bottom 96-well plates (Greiner Bio-One, Monroe, NC, USA) were coated with 100 μL of 25  μg/mL fetuin (Sigma-Aldrich, St. Louis, MO, USA) at 4 °C overnight. Influenza virus A/California/07/2009 was diluted in sample diluent (PBS, 1% BSA, 0.5% Tween-20) to an initial dilution of 1:10 and then serially diluted two-fold for 11 dilutions. A negative-control column was included containing 100 μL of only the sample diluent. Fetuin plates were washed three times with PBS-T (PBS  +  0.05% Tween 20). Next, 50 μL of serially diluted virus was added to the fetuin-coated plate containing 50 μL of the sample diluent in duplicate. Plates were incubated for 18 h at 37 °C and 5% CO₂. After incubation, plates were washed six times in PBS-T, and 100 μL of peanut agglutinin-HRPO (Sigma-Aldrich, St. Louis, MO, USA) diluted 1000-fold in conjugate diluent (PBS, 1% BSA) was added. Plates were incubated at RT for 2 h. Plates were washed three times in PBS-T, and 100 μL (500 μg/mL) of o-phenylenediamine dihydrochloride (OPD) (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-citrate buffer (Sigma-Aldrich, St. Louis, MO, USA) was added to the plates. Plates were incubated in the dark for 10 min at RT. The reaction was stopped with 100 μL of 1 N sulfuric acid. The absorbance was read at 490 nm on a BioTek plate reader (Agilent, Santa Clara, CA, USA). The dilution of the virus needed to achieve 90 to 95% NA activity was determined and used for subsequent NA inhibition ELLAs.