The Jio tariff revision that the company promised a week ago has become official, with the new or altered plans going live on October 19, the day of Diwali. Among the big changes is the introduction of the Rs. 459 pack that provides the benefits of Rs. 399 pack, but at 15 percent higher price. The Rs. 399 pack will provide less validity (and thus less data) to consumers, down from 84 days to 70 days. If you are looking for the Rs. 309 recharge, the validity is down from 56 days to 49 days. Another major change is that Jio users who exhaust their quota of daily high-speed Internet will now get Internet access at 64kbps speed, half of the 128kbps Jio offered till now.
Update: An earlier version of the article mentioned that Jio has axed the Rs. 309 plan. As it turns out, the plan has been removed from the list of Jio plans on its website, but it continues to be available a recharge option via the MyJio app. If this will continue to be the case in the future is unclear.
For postpaid users, the rude shock seems to be curtailed validity, as all plans now have a validity of 1 billing cycle (30 days) only. Before this tariff revision, Jio offered validities of up to three months with its postpaid plans. Moreover, there is a security deposit with each postpaid plan now, and post-FUP speed will be 64kbps.
Moving to the plans now, the Rs. 309 plan still remains in the postpaid listing, with 30GB data doled out at 1GB per day; the security deposit for the pack is Rs. 400. Next is the Rs. 409 pack, with Rs. 500 deposit and 20GB data without any daily restrictions on consumption. The Rs. 459 plan doesn’t seem to be an option for postpaid users.
Next up is the Rs. 509 Jio plan with Rs. 600 deposit and 2GB data per day, totalling 60GB. If you want 3GB daily data, there’s the new Rs. 799 pack with Rs. 950 security deposit. The most expensive postpaid plan is the Rs. 999 postpaid plan, which gives customers 60GB data without any daily limits and is available with security deposit of Rs. 1,150.
The Rs. 399 plan under the new tariff regime offers 1GB data and unlimited calls, free SMSes, and access to Jio apps, with a validity of 70 days. At this price, the company offered 84-day validity till now. Now it is the Rs. 459 recharge that offers 84-day validity, 1GB 4G data per day, and other freebies. The validity of the Rs. 509 plan has also been reduced from 56 days to 49 days, with high-speed data still capped at 2GB per day. For the Rs. 309 recharge, which is available only via the MyJio app or the recharge page on the website, you will get 1GB data per day with validity of 49 days, down from 56 days; interestingly, the Rs. 309 pack is not mention on the Jio website under the list of plans for prepaid users.
In high-value recharges, the data cap of the Rs. 999, Rs. 1,999, and Rs. 4,999 has been reduced as well, while their validities remain the same at 90 days, 180 days, and 360 days, respectively. The Rs. 999 recharge pack now offers 60GB data instead of 90GB, and Rs. 1,999 plan gives 125GB data, not 155GB. Similarly, Jio subscribers will get 350GB data with the Rs. 4,999 pack, down 30GB from the earlier 380GB. The Rs. 9,999 plan – the most expensive the company offered till now – seems to have been eliminated altogether.
As for the low-value packs, the Jio website mentions the Rs. 52 pack with 7-day validity and 1.05GB bundled data (150MB daily cap). Then there’s the Rs. 98 recharge plan that provides 150MB data per day for two weeks, totalling 2.1GB of 4G data. The company is providing just 70 and 140 free text messages (SMS) with these two packs, respectively. With the Rs. 149 pack, users will get 28-day validity, 4.2GB data (150MB per day), and 300 free SMSes. All calls remain free on the network across the prepaid pack.
Respiratory syncytial virus (RSV) is a primary cause of serious lower respiratory tract illness for which there is still no safe and effective vaccine available. Using reverse genetics, recombinant (r)RSV and an rRSV lacking the G gene (ΔG) were constructed based on a clinical RSV isolate (strain 98-25147-X).
Growth of both recombinant viruses was equivalent to that of wild type virus in Vero cells, but was reduced in human epithelial cells like Hep-2. Replication in cotton rat lungs could not be detected for ΔG, while rRSV was 100-fold attenuated compared to wild type virus. Upon single dose intranasal administration in cotton rats, both recombinant viruses developed high levels of neutralizing antibodies and conferred comparable long-lasting protection against RSV challenge; protection against replication in the lungs lasted at least 147 days and protection against pulmonary inflammation lasted at least 75 days.
Collectively, the data indicate that a single dose immunization with the highly attenuated ΔG as well as the attenuated rRSV conferred long term protection in the cotton rat against subsequent RSV challenge, without inducing vaccine enhanced pathology. Since ΔG is not likely to revert to a less attenuated phenotype, we plan to evaluate this deletion mutant further and to investigate its potential as a vaccine candidate against RSV infection.
Respiratory syncytial virus (RSV) is a non-segmented, negative-stranded RNA virus and a member of the Paramyxoviridae family. RSV is the most important cause of serious respiratory tract disease for young infants and children, but also for the elderly and immunocompromised persons. More than 50% of the children are infected within their first year of life. The clinical manifestations range from mild, common cold-like symptoms to more severe bronchiolitis and pneumonia. Clinical observations indicate that the first infection with RSV is generally the most severe, whereas subsequent infections tend to be increasingly milder [1]. The peak incidence of serious disease is at 2 - 6 months of age. RSV infection accounts for 40 - 45% of children hospitalized for bronchiolitis or lower respiratory tract disease [2]. The high disease burden indicates an urgent need for a vaccine against RSV, however there is currently no licensed vaccine available. One major obstacle to the vaccine development is the legacy of vaccine-enhanced disease in a clinical trial in the 1960s with a formalin-inactivated (FI) RSV vaccine. FI-RSV vaccinated children were not protected against natural infection and infected children experienced more severe illness than non-vaccinated children, including two deaths. Studies analyzing the RSV-specific immune response in mice indicate that both Th1 and Th2 CD4 T cell responses, as well as CD8 T cell responses can contribute to RSV vaccine-enhanced disease [3]. Lack of protection by FI-RSV is due to low and poorly neutralizing antibody responses and recently it was shown that low antibody avidity for protective epitopes was caused by poor Toll-like receptor stimulation [4].
Since the trial with the FI-RSV vaccine, various approaches to generate an RSV vaccine have been pursued without success. Attempts include classical live attenuated cold passaged or temperature sensitive mutant strains of RSV, (chimeric) protein subunit vaccines, peptide vaccines and RSV proteins expressed from recombinant viral vectors. Although some of these vaccines showed promising pre-clinical data, no vaccine has been licensed for human use due to safety concerns or lack of efficacy [5, 6].
Enhancement of RSV disease does not occur after natural RSV infection and has not been observed after inoculation with live attenuated vaccine candidates [7]. These are important facts in favor of a live attenuated RSV vaccine administered intranasally. The most challenging aspect of developing a live attenuated RSV vaccine is, however, to achieve an appropriate balance between attenuation and immunogenicity in the immunologically immature young infant who possesses varied levels of maternally acquired serum antibodies. Reverse genetics technology holds great promises for the development of such a live attenuated vaccine through its potential of including rationally designed, predetermined changes for vaccine candidates [8].
RSV expresses two major glycoproteins on its surface: the fusion (F) glycoprotein and the attachment glycoprotein (G). RSV F mediates penetration and syncytia formation, while RSV G is the putative attachment protein and is naturally expressed as a membrane-anchored and a secreted form [9]. The F protein is indispensable for virus replication and growth, whereas the G protein is not essential for virus growth in vitro [10]. RSV exists as two antigenic subgroups, A and B, with the greatest divergence occurring in RSV G (55% identity between the two RSV antigenic subgroups) [11]. While both F and G are important targets for antibody responses, F is considered to be the major stimulus for virus neutralizing antibodies.
A cold-passaged attenuated RSV subgroup B mutant (cp52) was found to replicate well in vitro despite the absence of functional G and SH proteins [12]. This vaccine candidate did neither replicate in chimpanzees nor induce neutralizing antibody response in human volunteers after intranasal administration, and was considered to be over-attenuated as a vaccine candidate. In the bovine model, however, a recombinant bovine RSV deletion mutant exclusively lacking the G gene was shown to protect calves against challenge virus replication [13]. A recombinant human RSV lacking the G gene has only been tested in the mouse model [14]. Thus, so far no immunization and challenge studies have been performed with RSV ΔG mutants in a more permissive animal model, such as the cotton rat (Sigmodon hispidus). This model has the advantage that RSV replicates well and vaccine-associated enhancement of disease has been well studied [15, 16].
Here we describe the construction of a recombinant human RSV (rRSV) and a deletion mutant lacking the G gene (ΔG), based on a recent RSV clinical isolate. These recombinant viruses were compared to wild type virus for replication in vitro and their efficacy as live-attenuated vaccine candidates was assessed in the cotton rat model.
Clinical isolate 98-25147-X (RSV-X), a RSV serogroup A strain, was used as the basis to create recombinant RSV by reverse genetics. Briefly, the RSV genome of RSV-X was PCR amplified into six large fragments, followed by sequential ligation (Figure 1A). A cDNA recombinant RSV mutant lacking the G gene was subsequently constructed by deletion of the G gene, including its gene-end and gene-start, from the full-length plasmid. To recover recombinant virus plasmids containing the full-length clones were transfected with helper plasmids driving the expression of RSV-A2 N, P, L and M2.1 proteins into MVA-T7 infected Hep-2 cells. To recover and amplify rescued virus, culture supernatants of the transfected Hep-2 cells were used to infect fresh Vero cells. Recovery of the rescued viruses was indicated by immunostaining of infected cells using a polyclonal antibody against RSV (data not shown). The rescued viruses were designated rRSV for the parental recombinant RSV and ΔG for the recombinant RSV mutant lacking the G gene. The identities of the recombinant viruses were verified by sequencing their viral RNA.
Generation of recombinant RSV-X virus. A) Schematic diagram of the RSV-X genome (genome length 15213 nt) and positions of the genetic tags inserted in the cDNA copy of the rRSV-X and ΔG constructs. The SexA I, Xma I, BssH II, Bsiw I, and Mlu I sites were introduced to facilitate construction. ΔG was recovered by excision of the fragment BssH II and BsiW I and subsequent religation of the vector. B) Expression of RSV proteins by rRSV and ΔG deletion recombinant viruses. Vero cells were infected at an m.o.i of 0.1 TCID50/ml. At 72 hr post infection cell monolayers was harvested and subjected to Western blotting using antiserum against RSV. The molecular weight size markers are depicted on the left and the position of the major RSV proteins are indicated at the right [29].
Expression of RSV proteins was verified by infecting Vero cells with wild type (wt) RSV-X, rRSV and ΔG followed by Western blotting of cell lysates (Figure 1B). A polyclonal antibody against RSV detected the major RSV structural proteins F, N, P and M in (r) RSV and ΔG infected cell lysates. In addition, as expected, the G protein was present in wt and rRSV, but not in ΔG infected cell lysates. Similarly, cells infected with ΔG did not show expression of the RSV G protein when immunostained with anti RSV-G antibodies (data not shown).
Replication of the recombinant viruses was characterized on Vero and Hep-2 cells (Figure 2). In Vero cells, growth kinetics of rRSV and ΔG were similar to that of the wild type virus. Peak titers of 107 TCID50.ml-1 were reached 72 hr post infection. Both recombinant viruses were attenuated on Hep-2 cells, as shown by a lower growth rate and 100-fold lower peak titers compared to the wild type virus. The ΔG virus did not form distinct syncytica, infection in Vero cells resulted in areas of rounded cells similarly as described by Teng et. al. [10](data not shown).
Growth of (recombinant) RSV in Vero and Hep-2 cells. Vero (A) and Hep-2 (B) cell monolayers were infected with wild type (wt) RSV, rRSV or ΔG with an MOI of 0.1 and incubated at 37°C. Cells were harvested at the indicated time points and virus TCID50 titers were determined in Vero cells.
We next ascertained the growth kinetics of the recombinant viruses on different epithelial and kidney cell lines including lung mucoepidermoid carcinoma cells (NCI-H292), human bronchial epithelial cells (16HBE140), human lung epithelial carcinoma cells (A549), and human kidney epithelial cells (293T). To this aim, the cell lines were infected with an MOI of 0.1 and the viral titers were determined at 72 h post infection (Figure 3). In all cell types examined rRSV and ΔG showed similar growth characteristics. Wild type virus reached titers of 106 to 107 in the A549 and Hep-2 cells, whereas the recombinant viruses produced approximately 100-fold lower titers. A similar picture was found on the NCI-H292 cells, although wild type virus only reached moderate titers of 105.2 and the recombinant viruses had 10-fold lower titers. All (r)RSV strains showed similar poor growth on the 293T cells with maximum titers of 104.3.
RSV replication in cell lines. Growth of RSV was tested in human lung mucoepidermoid carcinoma cells NCI-H292, human bronchial epithelial cell line 16HBE140, human lung epithelial carcinoma cells A549, human kidney epithelial cells 293T, human epithelial Hep-2 cells and monkey kidney Vero cells. Cells were infected with virus with an MOI of 0.1, harvested after 72 hr and virus CID50 titers were determined in Vero cells.
Wild type and recombinant RSV-X strains were compared with the extensively well characterized subtype A strain RSV-A2 for their ability to replicate in the cotton rat lung. Animals were infected intranasally (i.n.) and were sacrificed after three, five or seven days post inoculation. Lung tissues and nasal lavages were collected for virus titration in Vero cells. High amounts of virus were found for both RSV-X and RSV-A2 in the lungs and nasal washes on day three and five, reaching titers of 105; whereas low levels of virus could be isolated from the nose until day seven (Table 1). Replication of rRSV was 100-fold lower compared to wt RSV-X and reached maximum titers of approximately 102.8 in the lungs of half or three quart of the immunized animals, respectively at day three and five. At these days, most of the rRSV infected animals had low levels of virus in the nose. Replication of the ΔG mutant in the lungs and nose was below the limit of detection at any of the time points. Thus, although we did not observe a difference in replication kinetics between rRSV and ΔG in vitro, the absence of the G protein resulted in an attenuated phenotype of the latter virus in the cotton rat.
d The lower limit of detection for virus in the lungs and nose was respectively 2.1 log10TCID50 g-1 and 1.6 log10TCID50. Groups consisted of 4 animals. ND: not determined.
Pulmonary inflammation was assessed using a scoring scale system comparable to described by Prince [16]. Infection with either RSV-X or RSV-A2 induced a mild to moderate inflammation around peribronchi(oli), and in alveoli and bronchial mucous epithelium was moderately hypertrophied with a peak inflammation on day 5. Perivasculitis was sporadically seen and only in a minimal way. Infection pathology with rRSV was milder than wild type RSV-X. The ΔG mutant did not show any detectable inflammatory damage, which is in accordance with the inability to demonstrate replication of this virus in the lungs (data not shown). The wild type and viruses RSV-A2 or RSV-X, but not the recombinant viruses rRSV and ΔG, induced a minimal to slight modest infiltration of eosinophils in the epithelium of the bronchus.
The recombinant viruses were subsequently examined for long term protective capacity against a pulmonary RSV challenge. To this aim, cotton rats were immunized with a single dose on day 0 and challenged on day 70 or 142. Five days after challenge infection, on day 75 or 147, respectively, the animals were sacrificed. A single immunization with either rRSV or ΔG induced sufficient immunity to protect the animals completely from challenge virus replication in the lungs up to 147 days after immunization (Table 2). rRSV immunization could protect the majority of animals against challenge virus replication in the nose, whereas ΔG could not. Both recombinant RSV-X viruses induced high serum RSV antibody titers at day 70, which dropped moderately at day 142. Furthermore, protection from pulmonary inflammation could be demonstrated until day 75 for both rRSV and ΔG (Figure 4). At this time, protection was significant for all histopathological parameters. Neither rRSV nor ΔG could fully protect against challenge histopathology at day 147.
a Cotton rats were infected with 105 TCID50 of the indicated virus at day 0. Groups consisted of 6 animals.
b At the indicated days cotton rats were challenge i.n. with 106 TCID50 of RSV-X, and sacrificed 5 days post challenge.
c Lungs and nasal washes were harvested and virus titers were determined. The lower limit of detection for virus in the lungs was 2.1 log10TCID50 g-1 and in the nose 1.6 log10TCID50.
d At the indicated days sera were collected and the neutralizing antibody titer against RSV-X was determined. The pre-infection serum titers were <3.3 (reciprocal log2) for all animals in the study.
Long term protection against RSV challenge lung histopathology in cotton rats. Cotton rats were immunized i.n. at day 0 with 105 TCID50 rRSV or ΔG. Challenge was performed at day 70 and 142 with 106 TCID50 RSV-X (i.n) and the animals were sacrificed 5 days later, at day 75 and 147, respectively. Groups consisted of 6 animals. Mean histopathological scores of following histopathological parameters: peribronchiolitis (black bars), hypertrophied mucous cells (brown bars), peribronchitis (white bars) and alveolitis (gray bars). Ctrl: control animals; mock: mock infected, challenged animals. *: statistically significant different (P < 0.05) compared to mock infected group based on the Wilxocon test.
This report describes that a recombinant RSV lacking the G protein (ΔG) is able to induce long-lasting protection against RSV challenge infection in cotton rats. A clinical isolate from 1998 from the Netherlands was used as source material for construction of the recombinant RSV. Compared to the parental recombinant rRSV, this ΔG virus was highly attenuated since replication of this virus in the cotton rat lungs could not be detected. Although attenuated, this virus could induce long-term protection against both wild type RSV challenge infection and the occurrence of infection-induced lung pathology.
This is the first description of a recombinant human RSV based on a clinical isolate. So far, the described recombinant RSVs have been based on the laboratory adapted A2 strain. These have been extensively studied, both in vitro and in vivo [8]. RSV-A2 was originally isolated in the 1960-s, while RSV-X is a clinical isolate from 1998. Although the growth characteristics of wild type RSV-X and its derived recombinant viruses were comparable in kidney epithelial cell lines like Vero and 293T, the recombinant viruses were attenuated in human epithelial cell lines like Hep-2, A549 and 16HBE140. Attenuation of recombinant RSV on Hep-2 cells has been reported in other studies [14, 17] and is postulated to be a general feature of clonal RSV from one genetic sequence. Alternatively, to incorporate restriction endonuclease sites for cloning purposes, the intergenic regions of the RSV genome of the RSV-X clone differ 20 nucleotides from the wild type sequence. This might influence virus transcriptional regulation and explain the attenuated phenotype of the recombinant viruses observed in human epithelial cells [18].
A high and comparable level of virus titers in cotton rat lungs and nasal washes was observed for both RSV-X and RSV-A2, with peak titers occurring on day five after infection, similar as described previously [16]. In addition, both virus strains induced similar infection pathology in the lungs. In contrast, replication of recombinant RSV-X was 100-fold attenuated compared to wt RSV-X. Deletion of the G gene from recombinant RSV-X lacking the G protein abrogated its detectable replicative capacity in vivo. Thus, although we did not observe a difference between rRSV and ΔG in vitro, the absence of the G protein clearly attenuated the ΔG virus in the cotton rat lungs and nose.
Single immunization of cotton rats with the attenuated rRSV or ΔG viruses conferred long-term protection (147 days) against challenge RSV replication in the lungs and induced high titers of RSV serum neutralizing antibodies, although these antibodies wane in time. Although no virus could be detected in the lungs after immunization, low levels of virus could be detected in the nasal washes, especially with the ΔG virus. Both ΔG and rRSV conferred protection against pulmonary inflammation which lasted at least until day 75. At this day, there was no difference in lung inflammation between rRSV and ΔG immunized or uninfected animals, confirming that immunization with live attenuated RSV is not associated with RSV enhanced disease as occurs with FI-RSV vaccines. Protection against infection pathology waned sooner than protection against challenge virus replication. Mild inflammatory responses in the lungs were detected in animals immune to RSV, even in the absence of detectable virus replication. The detection levels of RSV lung titers might not have been sensitive enough to detect these low levels of RSV replication, since low levels of virus could be detected in the nasal washes.
The observed level of attenuation of the ΔG virus is consistent with previously reported data on cp52, a cold passaged RSV mutant lacking functional G and SH. This mutant was highly attenuated in cotton rats, attenuated and immunogenic in chimpanzees, but was found to be overattenuated for RSV-seronegative infants and children [19]. However, this mutant possesses in addition to the deletion of functional G and SH, an aberrant SH-G intergenic region and mutations in the F and L gene. A number of recombinant RSV-A2 have been made that lack either the complete G gene or express truncated, secreted or membrane forms of G, but only a recombinant RSV-A2 with the membrane form of G has been tested for its protective efficacy in BALB/c mice [20]; but this mutant was considered to be over-attenuated. However, the attenuated nature of this mutant is not convincing; other papers describe that such a recombinant RSV-A2 showed enhanced virulence compared to wild type RSV-A2 [21, 22].
A recombinant bRSV lacking the G gene induced bRSV neutralizing antibodies after intranasal immunization of calves. Although it was not clear whether this mutant was able to replicate in the lungs, calves were protected against a subsequent bRSV challenge infection after mucosal administration [13]. Human metapneumovirus (HMPV) belongs to the same subfamily of the Pneumovirinae as RSV, recombinant viruses lacking the G gene are replication competent in the upper and lower respiratory tract of hamsters and were attenuated compared to wt HMPV. Intranasal immunization of HMPV lacking the G gene conferred protection against challenge virus replication in the lungs but not in the nasal turbinates [23]. These results are in agreement with our results. More importantly, we have shown that this highly attenuated ΔG confers long-term protection against subsequent challenge.
Deletion and/or functional inactivation of the gene coding for the G protein prevents a number of problems and complications associated with potential RSV vaccine candidates. One purpose is vaccine safety: RSV without G is highly attenuated in its host because it will not be able to infect host cells efficiently [13, 19], is not likely to revert to a less attenuated phenotype and does not show enhanced disease. Moreover, a substantial role for the G protein has been suggested in the induction of undesired immunological responses, including enhanced immune pathology [24] and possible skewing of the immune system towards an allergy (and asthma) prone state under certain genetic predispositions [25]. In contrast, several recent studies have shown that G is not implicated in vaccine enhanced disease induced by immunization with formalin-inactivated RSV [26]. Nevertheless, candidate RSV vaccines will have to prove they will not induce enhanced disease in vaccinees.
Our data represent the first characterization in a relevant animal model of a recombinant RSV lacking the G gene that can be considered safe because of it is high attenuation profile in vivo (no detectable virus replication in the cotton rat lungs and nasal washes) and its lack of induction of enhanced disease in the cotton rat model. Moreover, it has the capacity to induce long lasting protection against challenge virus replication. This study demonstrates that the attenuated recombinant RSV and the highly attenuated recombinant RSV virus lacking the G gene confer long-term protection against challenge virus replication and inflammation after a single immunization. The RSV lacking the G gene is not likely to revert to a less-attenuated phenotype, we plan to evaluate this mutant further to investigate its potential as a vaccine candidate against RSV infection.
Monkey kidney Vero cells (CCL-8, American Type Culture Collection (ATCC)) were cultured (37°C, 5% CO2) in M199 medium (Invitrogen) supplemented with heat-inactivated 5% fetal bovine serum (FBS, Hyclone) and PSG (100 units of penicillin, 10 μg of streptomycin and 292 μg of L-glutamine/ml, Invitrogen). Hep-2 (CCL-23, ATCC) human lung epithelial carcinoma cells A549 (CCL-185, ATCC) and human kidney epithelial cells 293T (CRL-11268, ATCC) lung mucoepidermoid carcinoma cells NCI-H292 (CRL-1848, ATCC), human bronchial epithelial cell line 16HBE14o [27] were cultured in DMEM medium (Invitrogen) with 10% FBS and PSG. RSV infected cells were grown at 37°C in DMEM medium supplemented with 1% FCS and PSG. The following RSV strains were used: A2 (ATCC, VR1302) and clinical isolate 98-25147-X (Leiden University Medical Centre, The Netherlands). The latter virus was isolated in 1998 and propagated for 9 passages on Hep-2 cells (CCL-23, ATCC). This stock was designated as RSV-X [Genbank FJ948820] and was used for generation of cDNA. RSV-X virus was determined as a subtype A antigenic isolate and genotyped as a GA2 virus. Modified vaccinia virus Ankara expressing T7 RNA polymerase (MVA-T7) was kindly provided by G. Sutter (Paul-Ehrlich Institute, Langen, Germany).
The antigenomic cDNA spanning the entire RSV-X strain genome was assembled into a single molecule by sequential ligation of RSV X cDNA fragments (Figure 1). This was facilitated by sequential cloning six cDNA fragments of the RSV genome into one expression vector to create a complete full length cDNA. Each fragment was sequenced completely using an ABI 310 DNA sequencer (Applied Biosystems). Five restriction sites (SexA I, Xma I, Bswi I, BssH II and Mlu I) were artificially introduced in the intergenic regions during the cloning procedure to help in cloning as well as to serve as markers to confirm the identity of the recovered recombinant virus. cDNA fragments were generated by reverse transcriptase (RT) PCR performed with Thermoscript reverse transcriptase (Invitrogen) and High fidelity platinum Taq polymerase (Invitrogen) using RSV-specific primers based on the sequence of RSV-A2 (primer sequences are available upon request). The first cDNA fragment had a T7 RNA polymerase promoter sequence located immediately proceeding nucleotide (nt) 1 and encompassed the leader sequence, NS1, NS2 and had an engineered SexA I site at nt position 1246. The second fragment encompassed N (nt 1246 to 2467) and was flanked by restriction sites SexA I and Xma I. The third fragment encompassed P, M, SH (nt 2467 to 4800) and was flanked by restriction sites Xma I and Bswi I. The fourth fragment encompassed G (nt 4800 to 5730) and was flanked by restriction sites Bswi I and BssH II. The fifth fragment encompassed F, M2.1 M2.2 (nt 5730 to 8587) and was flanked by restriction sites BssH II and Mlu I. The sixth fragment encompassed L, the trailer sequence (nt 8587 to 15213), the hepatitis delta virus ribozyme followed by a terminator of the T7 polymerase and was flanked by restriction sites Mlu I and Kpn I. In addition to the construction of a full length recombinant RSV-X cDNA clone (pRSV-X), a recombinant RSV-X cDNA clone lacking the G gene was recovered from the full length clone by excision of the BssH II and BsiW I fragment, followed by religation (pRSV-XΔG). The full length cDNA clones were verified by sequencing completely.
Recombinant RSVs were recovered from cDNA largely as described before [17, 28]. MVA-T7 infected Hep-2 cells were transfected with the antigenic plasmid (pRSV-XΔG or pRSV-X) and a set of four helper plasmids expressing the RSV N, P and M2.1 proteins (designated pcDNA6-A2-N, pcDNA3-A2-P, pcDNA6-A2-L, and pcDNA6-A2-M2.1 respectively). These helper plasmids expressed RSV genes of RSV strain A2. The amounts of the plasmids added was as follows: 1.6 μg pRSV-XΔG or pRSV-X, 1.6 μg pcDNA6-A2-N, 1.2 μg pcDNA3-A2-P, 0.4 μg pcDNA6-A2-L, 0.8 μg pcDNA6-A2-M2.1. After 3 - 4 hrs of incubation at 32°C, 500 μl of Optimem (Invitrogen) with 2% FCS was added and the cells were incubated at 32°C for 3 days. Cells were then scraped and the mixture of scraped cells and medium containing the rescued virus was used to infect fresh cultures of Vero cells grown in DMEM + 1% FCS + PSG. The latter procedure was repeated for 4 - 5 times to amplify rescued virus. The viruses were purified and concentrated by PEG precipitation of culture supernatants and stored as stocks at -80°C.
Expression of the RSV proteins in infected Vero monolayers was confirmed by immunostaining with an anti-RSV goat polyclonal antibody (Biodesign), a monoclonal antibody against RSV-G (Mab 131-2G, Chemicon or Mab L9), or a monoclonal antibody against RSV-F (130-8F, Chemicon). Vero cells were infected at an m.o.i. of 0.1 TCID50/ml. At 72 hr post infection cell monolayers were harvested and approximately 5 × 104 cell equivalent was subjected to electrophoresis on a 12% SDS-polyacrylamide gel (Pierce), transferred to optitran BA83 nitrocellulose membrane (Whatman) and subjected to Western blotting using a mixture of Mab L9, Mab 130-8F and polyclonal RSV antibody. In addition, the identities of the recombinant viruses were verified by sequencing viral RNA of the rescued viruses. Growth analysis of recombinant viruses was determined by infecting 50 - 60% subconfluent Vero or Hep-2 cells with at MOI of 0.1. The infected monolayers were incubated at 37°C. At 0, 24, 48, 72, 96, and 120 hr post infection, cells and media were harvested and stored at -80°C.
Virus titers were determined by 50% tissue culture infective dose (TCID50). TCID50 assays were first visually inspected for cytopathic effect after 7 days incubation at 37°C. The supernatants were subsequently analyzed by antigen capture ELISA using goat polyclonal antibody against RSV. To determine RSV titers in the lungs of cotton rats, the right lungs were removed, weighed, homogenized in stabilizing buffer, and stored at -80°C. RSV lung titers were determined on Vero cells and expressed in log10TCID50 per gram lung; the lowest limit of detection in cotton rat lungs was 2.1 log10 TCID50.g-1. To determine RSV titers in the nose, nasal washes were obtained by flushing the upper trachea with 2 ml PBS with 7.5% sucrose.
Two-fold serial dilutions starting at 1:10 of cotton rat serum were prepared in virus diluent (DMEM supplemented with 1% FCS and PSG). Each serum was mixed with an equal volume of virus (50-100 plaques/well) and incubated for 1 hr at 37°C. Vero monolayers, prepared in 96-well plates, were infected with 50 μl/well (in triplicate) of the serum/virus mixture. After centrifugation for 1 h at 700 × g and incubation of 1 hr at 37°C, supernatant was removed and cells were overlaid with 1.0% methyl cellulose prepared in DMEM supplemented with 1% FCS and PSG. After 2 days at 37°C, the overlay was removed and the cells were fixed with 80% acetone and stained with polycolonal anti-RSV HRP. Plaques were counted and plaque reduction was calculated by regression analysis to provide a 60% plaque reduction titer.
Cotton rats (Sigmodon hispidus) were originally obtained from Charles River Laboratories (Netherlands) and used for establishment of an in-house specific pathogen free breed. Unless otherwise specified, young (4 to 8 weeks) adult cotton rats were intranasally (i.n.) immunized at day 0 with 100 μl 105 TCID50 RSV-X, rRSV, or ΔG virus preparations. Each group consisted of 4 - 6 animals. At indicated time points the animals were challenged i.n. with 100 μl 106 TCID50 RSV-X. Mock immunized animals received PEG precipitated supernatants of mock infected Vero cells. Control animals were mock immunized and mock challenged. All infections were administered under anesthesia.
The left lung was inflated intratracheally with 10% neutral buffered formalin and fixed for at least 24 hours. Lung tissue was longitudinal embedded in paraplast, haematoxilin and eosin (HE) stained sections (5 μm), in which all air passages from bronchi to terminal bronchioles and alveolar ducts were present, were judged light microscopically. A whole section of a lung was considered in the evaluation of the following histopathological parameters: hypertrophy of bronchial (mucous) epithelium, the presence of subepithelial inflammatory cells around bronchi (peribronchitis), bronchioles (peribronchiolitis) and blood vessels (perivasculitis) and in the alveoli (alveolitis), comparable to Prince et al. [16]. In addition the presence of intra-epithelial eosinophils was scored in the bronchus. These histopathological parameters were each semi quantitatively scored in a blinded manner by a pathologist as 0 = absent, 1 = minimal, 2 = slight, 3 = moderate, 4 = strong, and 5 = severe respectively. In this score, the frequency as well as the severity of the lesions was incorporated. For each group the mean score of each histopathological parameter was calculated. Since the scoring was non-linear, the histological data were analyzed by using the nonparametric Wilxocon test. Results for which the P value was <0.05 was considered to be significant.
We thank E. Walsh for monoclonal L9, J. Robinson and L. van der Ven for their assistance in performing these studies, D. Elberts, P. van Schaijk and C. Soputan for excellent biotechnical support and W. Huisman for critical reviewing this manuscript.
MW participated in design and interpretation of the experiments, performed the research, and wrote the manuscript. JB carried out the virological experiments. MVB performed cloning experiments. YVR performed virus neutralization assays. PJMR carried out the pathological analysis and helped draft the manuscript. WL conceived the study and was involved in drafting the manuscript. All authors approved the final version of the manuscript.
Bioinformatics and evolutionary insight on the spike glycoprotein gene of QX-like and Massachusetts strains of infectious bronchitis virus
Infectious bronchitis virus (IBV) is a Gammacoronavirus of the family Coronaviridae and is a causative agent of an economically important disease in poultry. The spike glycoprotein of IBV is essential for host cell attachment, neutralization, and is involved in the induction of protective immunity. Previously obtained sequence data of the spike gene of IBV QX-like and Massachusetts strains were subjected to bioinformatics analysis.
On analysis of potential phosphorylation sites, the Ser542 and Ser563 sites were not present in Massachusetts strains, while QX-like isolates did not have the Ser534 site. Massachusetts and QX-like strains showed different cleavage site motifs. The N-glycosylation sites ASN-XAA-SER/THR-55, 147, 200 and 545 were additionally present in QX-like strains. The leucine-rich repeat regions in Massachusetts strains consisted of stretches of 63 to 69 amino acids, while in the QX-like strains they contained 59 amino acids in length. An additional palmitoylation site was observed in CK/SWE/082066/2010 a QX-like strain. Primary structure data showed difference in the physical properties and hydrophobic nature of both genotypes. The comparison of secondary structures revealed no new structural domains in the genotypic variants. The phylogenetic analyses based on avian and mammalian coronaviruses showed the analysed IBV as closely related to turkey coronaviruses and distantly related to thrush and munia coronaviruses.
The study demonstrated that spike glycoprotein of the Massachusetts and the QX-like variants of IBV are molecularly distinct and that this may reflect in differences in the behavior of these viruses in vivo.
Infectious bronchitis virus (IBV) causes avian infectious bronchitis, a highly contagious disease that affects poultry and produces severe economic losses worldwide. IBV belongs to the order of Nidovirales, family Coronaviridae and to genus of Gamma-coronavirus group 3 [1]. The genome is positive-sense single stranded RNA of about 27.6 kb and contains 5′ and 3′ untranslated regions [2–4].
The Massachusetts strain was initially isolated in 1940 in USA and clinically characterized as causing respiratory signs and decrease in egg production. It is widely distributed and predominant in many countries around the world [5]. The Massachusetts strain can infect different organs and some of the isolates could be recovered from gastrointestinal tract especially from the cecal tonsils [6, 7]. First IBV QX infections characterized by swelling of stomach in chicken flocks were reported in Qingdao China in 1996 [8]. Following the years the IBV QX viral infections was associated to either proventriculitis or renal infections in IBV vaccinated flocks in China [9, 10]. The decreases in egg production and false layers in mature hens in chicken flocks were also reported with QX infections [10, 11]. Later on Chinese QX of IBV was isolated from a backyard flock in Russia where the prevalence of Chinese QX-like genotype was common [12, 13]. Subsequently there have been increasing reports of QX-like cases in Europe, where the involved virus bearded sequence similarities to QX-like viruses, such as the cases in Belgium, Denmark, France, Hungry, Germany, The Netherlands, Poland, Russia, Slovenia, Spain, Sweden, and UK [7, 14–19]. These IBV viruses were termed as European QX-like viruses symptoms associated with bad egg quality, false layers in mature hens [2].
The spike glycoprotein of the virus is translated as a pre-cursor protein (SO) that is later cleaved into the N-terminal S1 and C-terminal S2 glycopolypeptides. The spike gene is highly variable, especially the S1 part, due to insertions, deletions, substitutions and recombination events [14, 20, 21]. The S1 part of the spike glycoprotein contains serotype specific virus neutralizing epitopes and is responsible for the hemagglutinating activity and for infectivity [22]. Due to this variability in nucleotide sequences, the cross protection between serotypes is low. Changes as little as 5% in the S1 sub-unit have been able to alter the protection ability of a vaccine [23]. The spike glycoprotein cleavage site motif has been described in bovine coronavirus, turkey coronavirus and human coronavirus, however, it is not found in all coronaviruses [24]. The cleavage recognition site in the spike glycoprotein of IBV consists of five basic amino acids, which is cleaved by host cell serine proteases [25]. Serine proteases are hydrolases that cleave peptide bonds [26, 27]. The cleavage recognition site sequence is not involved in pathogenicity because attenuated and pathogenic strains (same virus) possess identical cleavage recognition site sequences [26]. The spike glycoprotein of IBV is involved in induction of protective immunity, neutralization and attachment to the host cell [28, 29]. It contains crucial virus neutralizing epitopes and serotypic specific sequences that are involved in, induction of protective immunity, tissue tropism and attachment into host cells [22, 25, 30–32].
Palmitoylation is essential for the screening, localization and trafficking at sub-cellular level, protein-protein interactions, and to pre-determine functional properties of proteins [33, 34]. N-glycosylation properties of the glycoprotein were shown to be involved in changes of virulence and cellular tropism in lactate dehydrogenase-elevating virus [35]. Protein phosphorylation has key role in regulation of physiological functions in the cells of prokaryotes and eukaryotes [36]. Leucine-rich repeat (LRR) is a structural motif of the protein like a alpha/beta horseshoe fold which is relevant for protein-protein interactions [37, 38]. In this study, palmitoylation, leucine-rich repeat and N-glycosylation characteristics of IBV Massachusetts and QX-like strains were evaluated using bioinformatics tools in order to determine the extent of difference in structural and functional features between the strains.
The objective of the present study was to analyze the molecular characteristics of selected isolates belonging to classical (Massachusetts) and emerging (QX-like) genotypes of IBV to indentify differences in potential functional motifs of the spike glycoprotein, predicted by bioinformatics analysis. In addition an evolution analysis with regards to avian and mammalian coronavirus was performed.
The loss or acquisitions of N-glycosylation sites in the spike protein were predicted. Thirty N-glycosylation sites were found in the Massachusetts strains, while 35 N-glycosylation sites were present in the QX-like strains. The N-glycosylation sites ASN-XAA-SER/THR-55, 147, 200 and 545 were additionally present in spike gene of QX-like strains. The results revealed that most of the N-glycosylation sites were conserved within genotype in the analyzed strains, except in strain CK/SWE/082066/10 that had lost ASN-XAA-SER/THR-533.
The analyses revealed that there were different in number and location of potential phosphorlated peptides in the spike glycoprotein. In Massachusetts isolates phosphorylation sites at position Ser54, Ser79, Ser312, Ser404, Ser534, Ser618, Ser685, Ser908 and Ser1061 were found to be conserved. While two isolates CK/SWE/242/95 and CK/SWE/748/95 that did not contain phosphorylation sites at position Ser542 and Ser563. In contrast, they contained additional potential phosphorylation sites at Ser177 and Ser878. QX-like strains did not contain a potential phosphorylation site at position Ser534 and acquired a new potential phosphorylation site at position Ser513.
The cleavage site motifs of analyzed strains are presented in Table 1. Massachusetts and QX-like strains showed a different cleavage site motif. From the analyzed strains of both genotypes, six strains have shown the cleavage site at amino acid position 537 arginine, while two strains CK/SWE/242/95 and CK/SWE/748/95 presented cleavage site at histidine.
Leucine-rich repeat (LRR) regions were observed in the spike glycoprotein consisting of 59 to 69 amino acids in length. In isolate CK/SWE/748/95 two LRR regions were found, starting at position at 771 (ATQLQARNNAL) and 1147 (LEKLSILKTYI), respectively. The LRR regions in Massachusetts strains consisted of stretches of 63 to 69 amino acids, while in the QX-like strains they contained 59 amino acids in length.
The data regarding the primary structures are presented in (Additional file 1: Table S1). The strains of Massachusetts group were found to have differences of approximately 0.3-1.2 KDa in their molecular weights, while QX-like strains possessed similar molecular weight. The isoelectric points (pI) 5.63-6.72 of the Massachusetts strains were comparatively higher than pI 5.73 of QX-like strains. There were 82-87 positively charged residues (Arg + Lys) in the QX-like strains, 2-4 residues more than Massachusetts strains. The computed instability index and aliphatic index (AI) showed variable values (83.79-85.91) in all analyzed strains. The grand hydropathicity (GRAVY) was found to have negative values in all strains. The primary structure data showed difference in the hydrophobic nature of both genotypes. The primary structures of the spike glycoprotein were found to be relatively variable in the strains.
The data regarding the secondary structures are presented in (Additional file 2: Table S2). All the analyzed isolates contained 18 to 19 alpha helices, 47-58 beta sheets and 63-73 residual coils. In the case of alpha helices and beta sheets, there was additionally one alpha helix and 11 beta strands in Massachusetts strains than in QX-like strains.
The phylogenetic analysis of the complete spike gene based on avian coronaviruses revealed the sequences of the analysed strains segregated into three main groups (Figure 1). Group I was composed of IBV sequences that clustered together with those of Massachusetts, Arkansas and QX-like genotypes, as previously described by Abro et al. (2012) [14]. The definition of those genotypes is supported by high bootstrap values of 100%. Group II consisted of turkey coronaviruses branching closely to the IBV. Group III consisted of sequences of munia and thrush coronaviruses, branching into two clusters: one comprising munia coronaviruses and the other thrush coronaviruses. The high bootstrap values (100%) indicate a true distance between these clusters and that they are likely species-specific.
Phylogenetic analysis based on complete spike gene of avian coronaviruses showing the relationship between IBV and avian coronaviruses. The isolates analysed in this study are indicated by square.
The phylogenetic analysis based on the spike glycoprotein of avian and mammalian coronaviruses strains has shown as the following discrimination (Figure 2): a) Sequences of IBV and turkey coronaviruses making the majority of avian coronaviruses. The sequences of Beluga whale coronaviruses and one bat-coronavirus HKU2 were distantly related to this branch of avian sequences, altogather composing the gama-coronaviruses. b) A set of sequences comprising separate clusters of avian, bat, human and feline coronaviruses, thus including both avian and mammalian coronaviruses. c) A set of strictly mammalian coronaviruses, branching into the SARS, human coronavirus HKU1, bat coronaviruses and a cluster majorly of animal coronaviruses comprising murine, equine, porcine and bovine coronaviruses. The sequence of human coronavirus OC43 was found in this cluster, curiously identical to a coronavirus identified in pigs and closely related to bovine coronaviruses. Porcine hemagglutinating encephalomyelitis virus occupied a distinct place between the human and equine coronaviruses clusters.
Phylogenetic analysis based on complete spike gene of avian and mammalian coronaviruses showing the relationship between coronaviruses in different host species.
A genetic analysis of the complete spike glycoprotein of selected isolates of Massachustes and QX-like strains was carried out in order to predict molecular features by application of molecular and In silico technologies.
In the isolate CK/SWE/082066/10, amino acid phenylalanine substituted asparagine resulting in the loss of a potential N-glycosylation site at position 533. The five additional N-glycosylation sites found in QX strains may influence cellular tropism. The variation in N-glycosylation sites may affect survival and transnsmission of the virus. The small changes can lead to disturbance in folding and conformation of the molecule. The variations in N-glycosylation sites may affect the interaction with receptors and render a virus more susceptible to host innate immune system and lower recognition by antibody, hence affecting the virus replication and infectivity [39–42]. In a virus belonging to the Coronaviridae, lactate dehydrogenase elevating virus (LDEV), the loss or acquisition of N-glycosylation sites in the protein has been reported to determine changes of virulence and cellular tropism [35].
The presence of serine, threonine and tyrosine residues, with the right context for potential phosphorylation was investigated in the spike glycoprotein. The results have shown that the spike glycoprotein of IBV genotypes contained serine and thereonine peptides with potential for phosphorylation. The substitution of a serine by amino acid alanine at positions 563 caused loss of a potential phosphorylated peptide in strains CK/SWE/242/95 and CK/SWE/748/95. Wilbur et al. (1986) reported that N protein is the only structural protein that contained phosphorylation activity in coronaviruses [43]. In this context we have observed potential phosphorylated peptides in the spike glycoprotein of IBV; however, this seems to be of no consequence for phosphorylation, since this property has not been identified in the spike which is well established as a glycoprotein.
The difference in cleavage site motifs of the spike glycoprotein of both genotypes, seem to indicate a correlation with geographical distribution of strains. It has been reported that serotypically and genotypically different IBV contain distinct cleavage site in the spike protein. However, the cleavage site sequence did not correlate with pathogenicity by comparing cleavage recognition sites in pathogenic and attenuated strains of IBV [26]. The cleavage site motifs determined in Massachusetts and QX-like strains in present study are different from previously reported by Jackwood et al. (2001) [26]. The replacement of arginine with histidine at amino acid position 537 in CK/SWE/748/95 and CK/SWE/748/95 may have influence antibody binding epitope. However, the functional meaning of this difference needs to be evaluated in proper biological assays. It has been reported that an IBV monoclonal antibody neutralization-resistant mutant (NR 18) had shown substitution of amino acid isoluecine for arginine in cleavage site motif which prevented cleavage and alter the conformation dependent monoclonal antibody binding epitope [26]. In orthomyxoviruses and paramyxoviruses more basic residues around the cleavage recognition site of the envelope glycoproteins enhance the cleavability by ubiquitous host cell proteases, a characteristic which is associated with host cell range and increased virulence [44, 45]. However, the relevance of cleavage site to virulence was not observed in case of IBV spike glycoprotein [26].
The data regarding leucine-rich repeat regions of isolate CK/SWE/748/95 demonstrated that two LRR regions may result in strong folding capacity in the spike glycoprotein. It has been reported that leucine-rich repeat (LRR) is a structural motif of protein like a alpha/beta horseshoe fold and is responsible for protein-protein interactions [37, 38].
An additional palmitoylation site was found only in isolate CK/SWE/082066/10, indicating that it is strain specific. However, experimental studies are required to identify the effect of additional palmitoylation in the virus biology. In influenza virus, palmitoylation of the cytoplasmic domain of the hemagglutinin has been shown to increase its association with lipid mass [46]. The deletion of palmitoylation sites in the hemagglutinin domain reduces hemagglutinin incorporation into the virion, thereby affecting the cellular trafficking [47]. It has been reported that removal of palmitoylation sites in HIV-1 virus effected in the formation of virus with low levels of glycoproteins incorporation and reduced infectivity [48]. Shen et al. (2009) reported attenuation of virus replication occurring due to mutations in the palmitoylation site in the non-structural protein nsp1 of Semliki Forest virus [49].
The predicted primary structures of the spike glycoprotein of both genotypic variants have shown a variable trend in physical properties such as isoelectric point, molecular weights and grand hydropathicity index. Grand average of hydropathicity index results suggested a hydrophobic nature of the spike glycoprotein in all variants. Cavanagh et al. (1986) reported that S2 sub-unit in N-terminal side of the membrane spanning hydrophobic domain of spike glycoprotein of IBV is susceptible to hydrolysis by many proteases [32].
The secondary structures prediction revealed that structures of the spike glycoprotein were found variable in both genotypic variants. It was also observed that there were no new structural domains and that no evolution has occurred in the structure of both genotypic variants. However, secondary structural variation in beta strands and residual coils have been observed between both genotypic strains.
A number of phylogenetic studies performed on the spike gene were mostly based on the partial sequences of S1 and N gene. Phylogenetic analyses based on the complete spike glycoprotein of avian coronaviruses demonstrated that IBV strains showed higher relatedness to turkey coronaviruses in comparison to other avian coronaviruses (Figure 1). This could be due to co-habitation of their host species. The appearance of a bat coronavirus branch close to these gammacoronaviruses suggests a possible common origin during earlier evolution of these viruses. The diversity of these viruses could have arisen from strong selective pressure resulting from mutations and recombinations that has been shown to occur in spike gene [14, 50, 51].
Phylogenetic analysis based on spike glycoprotein of avian coronaviruses and mammalian coronaviruses had revealed interesting trends. The mammalian beluga whale coronaviruses branched closer to the avian coronaviruses and two avian thrush and munia coronaviruses were related to the mammalian coronaviruses suggesting interspecies transmission. Furthermore, findings of the present study suggest that genetic diversity and molecular variation in sequences of spike glycoprotein of viruses made feasible the adaptation to new host species. Previously it has been reported that the RNA-dependant RNA polymerase gene and the whole genome of the beluga whale coronavirus was distantly related to IBV [52, 53]. Also a shift of tissue tropism in the virus has been reported [54, 55] resulting in infection of a wide range of avian host species especially those reared close to domestic fowl. For example, IBV cases were found in guinea fowl (Numida meleagris), Chinese peafowl (Pavo), teal (Anas) and partridge (Alectoris) [56].
The higher diversity in the avian, humans and bat-coronaviruses are due to continuous mutation, which may determine differences in tissue tropism. Previously it has been reported that diversity of coronaviruses due to a) infidelity of RNA-dependant RNA polymerase which lead to incorporation of nucleotide errors during replication [34, 57]; b) random template switching during RNA replication (copy choice mechanism) high rate of homologous RNA recombination events [4, 58]; c) extra plasticity nature in adjusting and modifying genes [59]. These factors were responsible for the generation and diversification of variants and adaption to new hosts and ecological niches in the coronaviruses [53, 59].
In silico predictions confirm previous data on phylogenetic analysis, showing that the spike glycoprotein of the Massachusetts and QX genotypes are molecularly different. Since the spike glycoprotein has important role in cell attachment, receptor recognition, the genetic diversity seen in this protein of IBV, avian, human, bat, and other animal coronaviruses may bear relation with host specificity. The identification of these characteristics will be foundation for the better understanding of epidemics, molecular mechanisms of infections, evolution and basis for development of effective vaccines. Bioinformatics approach is easy to perform, cheap and suitable to obtain predictions about functional motifs in a genome. However, the predicted findings require validation through proper laboratory experimentation.
The sequence data set generated as previously described by Abro et al. (2012) [14] was used for the analysis (Additional file 3: Table S3).
N-glycosylation sites were predicted by services available on http://www.cbs.dtu.dk/services/NetNGlyc. The potential phosphorylation sites were determined by using website http://www.cbs.dtu.dk/services/NetPhos. Leucine-rich repeat regions were determined by LRRfinder available at http://www.lrrfinder.com/result.php. Antigenic determinants were predicted with the maximum score with the high confidence for positive determinant motif in the amino acid sequence with the help of server http://mobyle.pasteur.fr. The primary structures of the spike glycoprotein were predicted by http://expasy.org/tools. Palmitoylation sites were predicted with the medium threshold frequency by using services at http://csspalm.biocuckoo.org/prediction.php. The secondary structures (alpha helices, beta strands and random coils) of the protein were predicted by using bioinformatics tools available on website http://npsa-pbil.ibcp.fr. The method of GOR4 http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html was used to identify the alpha helices, beta strands and coil residues. At least minimum three or more turns were taken in account for one helix, strand or coil in the structure of spike glycoprotein.
The comprehensive phylogenetic study was carried out based on the complete spike gene of avian and mammalian coronaviruses available in GenBank. The sequences included IBV Massachusetts type, M41, H120, Baudette, Arkansas, QX like strains and other sequences of Turkey coronavirus (TCoV), thrush coronavirus (ThCoV), munia coronavirus (MuCoV), human coronavirus (HCoV), SARS (Severe acute respiratory syndrome) coronavirus, bovine coronavirus (BCoV), equine coronavirus (ECoV), mouse hepatitis virus (MHV), murine coronavirus (MurCoV), feline coronavirus (FCoV), porcine epidemic diarrhea virus (PEDV), porcine respiratory coronavirus (PRCV), porcine hamagglutinating encephalalomylitis virus (PHEV), bat coronavirus and beluga whale coronavirus sequences (Additional file 3: Table S3; Additional file 4: Table S4). Multiple sequence alignment of complete spike gene was performed with CLUSTAL W [60]. All the gaps in the sequences created by insertions were deleted by using software Mega4 version 4 [61]. Phylogenetic trees were constructed by the Neighbour-joining method with the nucleotide substitution model of Kimura-2 parameter model. The tree reliability/robuststness of the hypothesis were evaluated by bootstrap of 1000 replicates. The data set was kept similar for the analysis. The Bayesian analyses were performed by using the software MrBayes 3.1 [62]. The model was set for the substitution rates lset nst = 6, rates of variation were performed by gamma distribution (rates = invgamma) posterior probability distribution ensured and 2000000 generation were run and standard deviation of split frequencies at .01. The output of the substitution model parameters were summarized with sump burnin = 250. The tree topology was prepared in the FigTree_v1.3.1. The trees were generated with various methods and the consensus tree was taken into account in this study.
12985_2012_1896_MOESM3_ESM.docx Additional file 3: Table S3: List of selected Swedish IBV strains and reference strains of avian coronaviruses of which sequences were downloaded from GenBank for the phylogenetic analysis in this study. The IBV strains analyzed in this study are indicated by star *. (DOCX 17 KB)
12985_2012_1896_MOESM4_ESM.docx Additional file 4: Table S4: List of reference strains of mammalian coronavirus sequences were downloaded from GenBank for the phylogenetic analysis in this study. (DOCX 17 KB)