# The first evidence of a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and unvaccinated broiler flocks in Algeria

Aziz Lounas, Karine Oumouna-Benachour, Hacène Medkour, Mustapha Oumouna

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Aziz Lounas, Karine Oumouna-Benachour, Hacène Medkour, Mustapha Oumouna. The first evidence of a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and unvaccinated broiler flocks in Algeria. Veterinary World, 2018, 11 (11), pp.1630-1636. 10.14202/vetworld.2018.1630-1636. hal-04922660

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# The first evidence of a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and unvaccinated broiler flocks in Algeria

A. Lounas¹, K. Oumouna-Benachour², H. Medkour³ and M. Oumouna²

1. Department of Medicine and Animal Surgery, Veterinary Sciences Institute, University of Blida, Algeria; 2. Department of natural sciences and life, Faculty of Sciences and Technology, University Yahia Fares of Medea, Algeria; 3. Microbes, Evolution, Phylogeny and Infection, UMR Aix-Marseille University, IRD, APHM, IHU Méditerranée Infection, Marseille, France.

**Corresponding author:** A. Lounas, e-mail: azizovet@yahoo.fr

**Co-authors:** KO: Benachourkarine1@gmail.com, HM: hacenevet1990@yahoo.fr, MO: oumouna@gmail.com

**Received:** 16-07-2018, **Accepted:** 12-10-2018, **Published online:** 29-11-2018

**doi:** 10.14202/vetworld.2018.1630-1636 **How to cite this article:** Lounas A, Oumouna-Benachour K, Medkour H, Oumouna M (2018) The first evidence of a new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and unvaccinated broiler flocks in Algeria, *Veterinary World*, 11(11): 1630-1636.

## Abstract

**Background and Aim:** Avian infectious bronchitis virus (IBV) frequently infects broilers and is responsible for severe economic losses to the poultry industry worldwide. It has also been associated with kidney damage in the broiler flocks. The aim of the present study is to determine the presence of IBV and its possible involvement in kidney damage of broiler chicks.

**Materials and Methods:** 14 clinically diseased broiler flocks from Western and Central Algeria were sampled and analyzed by hemagglutination inhibition (HI) test and reverse transcriptase-polymerase chain reaction (RT-PCR) followed by phylogenetic analysis.

**Results:** The QX (100%) and 4/91 (60%) IBV serotypes were the most prevalent in the kidney damaged broilers regardless of vaccination status. The molecular detection of avian IBV by RT-PCR identified six samples as positive, of which only two isolates were typable by sequencing. We identified a novel IBDZ13a genotype which showed 93% sequence homology to the partial-S1 gene sequence of the IB 4/91 commercial vaccine strain. Sequencing analysis characterized this virus as a novel and divergent IB 4/91 field virus with eight amino acid substitutions that might have resulted in altered immunogenicity.

**Conclusion:** The isolation of a new IBV strain (IBDZ13a) from vaccinated broiler flocks may explain the failure of the vaccination programs against IBV field strains. Combination of the HI test and RT-PCR indicated that the nephropathogenic IB outbreaks in broilers are related to this novel strain.

**Keywords:** avian infectious bronchitis virus, broilers, kidney damages, Algeria, hemagglutination inhibition test, reverse transcriptase-polymerase chain reaction, IBDZ13a.

## Introduction

Poultry production in Algeria faces many zootechnical and health constraints, such as viral infections like avian infectious bronchitis (IB). The avian IB virus (IBV), a member of the Coronaviridae family (order *Nidovirales* and genus *Coronavirus*), frequently infects broilers and egg-laying hens and leads to severe economic losses to the poultry industry [1]. Since its discovery in the 1930s [2], the IBV has been identified as the major cause of respiratory infections and poor zootechnical performances. Interestingly, it can also multiply in the renal tissue and cause nephritis [3], a phenomenon first described in the United States [4]. More recently, IBV-associated nephritis has been accepted as the most pressing problem in broiler flocks in many countries [5-9].

The most effective method of protecting poultry from IBV infections is through live or killed vaccines [1]. However, nephritis associated with infectious bronchitis has been observed in several vaccinated flocks, suggesting that the current vaccination strategies against IBV may not provide adequate protection [3,10]. In fact, outbreaks of IB are frequently caused due to the strains serologically different from those used for vaccination [11,12]. Since its discovery in 1931, a large number of serotypes or variants of IBV have emerged [5,13,14], and little or no cross-protection occurs between these serotypes [15]. Therefore, it is crucial to track epidemic-causing serotypes in each geographic region or country and produce new vaccines to control IB.

In Algeria, poultry flocks have been vaccinated against IB with the Massachusetts (Mass) strain combined with the IB 4/91 United Kingdom variant strain or 793/B since the past few years. However, kidney damage with suspicion of IB has also been reported in recent years in spite of vaccination but has not been confirmed so far. This has led to the speculation of the possible emergence of variant strains against

Copyright: Lounas, et al. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
which conventional vaccines are not completely
effective [16].

The aim of this study was to investigate the
presence of IBV among Algerian broiler flocks and
its possible involvement in broiler kidney damage.
Clinically diseased broiler flocks were sampled and
analyzed by the hemagglutination inhibition (HI) test
and reverse transcriptase-polymerase chain reaction
(RT-PCR) followed by phylogenetic analysis.

Materials and Methods

Ethical approval

All experimental procedures were approved by the
Institutional Animal Care Committee of the National
Administration of the Algerian Higher Education and
Scientific Research (Ethical approval number: 98-11,
Law of August 22, 1998) and were conducted accord-
ing to the recommendations of the “Guide for the Care
and Use of Laboratory Animals.”

Sample collection

Fourteen commercial broiler flocks from Western and Central Algeria in which the chickens were suffering from kidney damage and respiratory disease were sampled between January and December 2012. Trachea and kidney specimens were taken from 10 diseased chickens from each flock and were directly applied to the FTA® cards (GE Healthcare, UK) during the acute phase of the clinical outbreak. The samples were transported and analyzed at the Laboratory of Virology, Instituto Zooprofilattico Sperimentale Delle Venezie, Italy. In addition, blood samples were also collected from each bird to determine the prevalence of different IBV serotypes. The serum was collected from the samples by centrifugation (2000 rpm/min 5 min) and stored at -20°C for further analysis. HI test was performed at MSD International laboratory, Amman, Jordan.

RNA extraction from FTA® cards and reverse
transcription

Three pieces of 2 mm discs from the spotted area
of the filters were cut by a sterile scissor and placed
in 1.5 mL Eppendorf tubes. Separate sterile scissors
and pincers were used for each sample to prevent
cross-contamination. Each disc was placed in 750 µL
Trifast® FL (PEQLAB, Germany), homogenized, and
incubated for 5 min at RT. The Trifast FL kit (Peq
GOLD TriFast™ FL, Peqlab) was used to extract the
RNA from the FTA® paper according to the manufac-
turer's instructions (PEQLAB Biotechnologie GmbH,
Erlangen, Germany). RNA purity and concentration
were determined by NanoDrop® ND-1000 (Peqlab
Biotechnologie GmbH, Erlangen, Germany). Reverse
transcription was performed using the ImProm-II™
Reverse Transcription System (Promega, Madison, WI)
with random primers according to the manufacturer's
instructions. Briefly, 0.1 µM lyophilized oligonucle-
otides provided with the kit were dissolved in 1 mL
RNase-free water, and 1 µL of the random primer solu-
tion was mixed with 4 µL viral RNA and incubated

at 70°C for 5 min. After adding Buffer 5× (56 µL),
MgCl₂ (67.2 µL), dNTP (14 µL), RNAsin (7 µL),
ImProm II (14 µL), and H₂O (51.8 µL), the mixture
was incubated at 25°C for 5 min followed by 55°C
for 60 min. The reaction was terminated by heating at
70°C for 15 min and cooling on ice.

PCR amplification of the S1 gene

The primer sets (Oligo5 [forward]: 5'-TGA AAA CTG AAC AAA AGA CA-3' and CK2 [reverse]: 5'-CTC GAA TTC CNG TRT TRT AYT GRC A-3') were designed for the IBV S1 gene, which contains the hypervariable region 1 and most of the hypervariable region 2 [17]. The S1 gene was amplified with the Superscript® III One-Step RT-PCR Platinum® Taq HiFi (Invitrogen™ Life Technologies™, Carlsbad, CA), which generated a PCR product of 344 bp. For the PCR reaction, the following reaction mix was made: 35 µL Buffer 10×, 14 µL dNTP, 14 µL CK2, 14 µL Oligo5, 243.25 µL H₂O, 1.75 µL Taq polymerase (Taq HIFI, Invitrogen™ Life Technologies™, Carlsbad, CA), and 2 µL cDNA to a final volume of 25 µL. The PCR parameters were as follows: 45 cycles of denaturation (94°C, 20 s), annealing (51°C, 1 min), and polymerization (72°C, 1 min). The pre-denaturation step was done at 94°C for 5 min and post-polymerization step at 70°C for 10 min. A 20 µL aliquot of each PCR product was electrophoresed at 120 V for 40 min in 1.5% agarose gel, which was then stained with GelRed™ (Genaxxon Bioscience, Ulm, Germany, GmbH).

S1 gene sequencing

The amplified cDNA was purified using
the NucleoSpin® (MACHEHEREY-NAGEL GmbH,
Germany) Gel and PCR Clean-up kit according to the
manufacturer's instructions and sent for sequencing
to x-OVO Limited (x-OVO Limited Laboratories,
Thomson Cooper, United Kingdom). The nucleotide
and amino acid sequences of the S1 gene of the IBV
isolates were assembled, aligned, and compared with
reference IBV strains using BASIC BLAST program
in DNASTar. Phylogenetic analysis of the S1 amino
acid sequences was performed with the neighbor-join-
ing method using the DNASTar software [18]. The
partial S1 nucleotide sequences from the IBV iso-
lates have been submitted to the GenBank database
and assigned the accession number MG252734. The
nucleotide and amino acid sequences of the S1 gene
of IBV strains used for the comparison are available
in GenBank under the following accession num-
bers: CR 88 (KM067900), 4/91 (JN600614), Qx
(AF193423), Ma5 (AY561713), H120 (M21970),
M41 (GQ219712), D274 (KR106999), Italy 02
(AJ457137), and QX D388 (DQ674739).

HI test

The specific IBV strains that were used in this
study were M-41 (a US strain), 4/91 (European vac-
cine strain), D274 (a Dutch field isolate), and QX
(Chinese strain). HI test was performed using the
FLOKSCREEN IB-HI TEST kit according to the manufacturer's instructions. Briefly, the sera were serially diluted and mixed with an equivalent volume of the antigen. After 30 min of incubation at 20°C, the red blood cells of SPF chickens (anticoagulant solution) were added and incubated further for 40 min at 20°C. The plates were then tilted to observe inhibition of hemagglutination.

## Statistical analysis

Statistical Package for the Social Sciences version 20 (IBM, USA) was used for statistical analysis. The geometric mean titer (GMT) was calculated by counting the number of wells from all serum samples within one flock showing HI activity, and the average number was then cross-checked against GMT values given in Brug's table. The results were reported as the GMT for a given flock against a particular IBV antigen. Descriptive statistics including percentages, means, and frequency distribution were calculated for each of the variables.

## Results

Information regarding the sampling date, biosecurity status, region of sampling, vaccination programs, necropsic and clinical signs, the age of the broilers, and mortality rates are summarized in Table-1. We collected 14 samples from broiler farms located in Western and Central Algeria from the flocks showing kidney damage and high mortality rates. Eight of these farms had not vaccinated their flocks against IBV, 2 farms conducted the prototype vaccination program but with a short interval between the first and second vaccinations, and 4 farms vaccinated their broilers only against the Mass serotype. All vaccinations were performed through the drinking water route, and all the sampled broiler farms showed poor zootechnical performance with rare period elongation (until 71 days in farm 2) and high mortality rates (up to 50% in farms 11 and 13).

The seroprevalence of various IBV strains in broiler flocks suffering from kidney damage in Algeria is summarized in Table-2. All the broiler flocks (100%) tested in this study were seropositive for the QX variant strain, with high mean titer levels (MT=11 log₂) regardless of the vaccination status. Furthermore, 60% and 50% of the broiler flocks were seropositive for antibodies against the 4/91 and M-41 strains, respectively, although with low mean titer levels. The seroprevalence of IBV D-274 was the lowest with only 7% of the broiler flocks testing positive.

Six of the 14 broiler samples were positive for the IBV S1 gene, but only two isolates were typable by sequencing and showed a high load of IBV viral RNA (Ct₁=22.15 and Ct₂=31.4). One of the two sequences was identified as the IBDZ13a genotype. Furthermore, IBV was only detected in the kidney tissues and not in the trachea. All samples showed 93% homology to the partial-S1 sequence acquired from

<table><caption>Table-1: Farm characteristics and the history of IBV outbreaks in Algeria.</caption><thead><tr><th>Flock number</th><th>Age of sampling (d)</th><th>Season of sampling</th><th>Time between outbreak-sampling</th><th>Region</th><th>Biosecurity status</th><th>Mortality rate (%)</th><th>Clinical signs</th><th>Vaccination program</th></tr></thead><tbody><tr><td>1</td><td>51</td><td>Spring</td><td>20</td><td>West</td><td>Average</td><td>48</td><td>Renal, respiratory</td><td>No vaccination</td></tr><tr><td>2</td><td>71</td><td>Summer</td><td>23</td><td>West</td><td>Average</td><td>13</td><td>Renal, respiratory</td><td>d7: Ma 5</td></tr><tr><td>3</td><td>60</td><td>Summer</td><td>27</td><td>Central</td><td>Average</td><td>26.5</td><td>Renal, respiratory</td><td>d7: Ma5 d14: IB 4/91</td></tr><tr><td>4</td><td>56</td><td>Spring</td><td>16</td><td>Central</td><td>Average</td><td>15</td><td>Renal, respiratory</td><td>d5: Ma 5</td></tr><tr><td>5</td><td>52</td><td>Summer</td><td>14</td><td>West</td><td>Poor</td><td>30.5</td><td>Renal, respiratory</td><td>No vaccination</td></tr><tr><td>6</td><td>58</td><td>Winter</td><td>18</td><td>Central</td><td>Poor</td><td>22</td><td>Renal, respiratory</td><td>d5: Ma 5</td></tr><tr><td>7</td><td>65</td><td>Winter</td><td>25</td><td>Central</td><td>Average</td><td>25</td><td>Renal, respiratory, digestive</td><td>No vaccination</td></tr><tr><td>8</td><td>52</td><td>Winter</td><td>15</td><td>West</td><td>Poor</td><td>24</td><td>Renal, respiratory</td><td>No vaccination</td></tr><tr><td>9</td><td>42</td><td>Winter</td><td>11</td><td>Central</td><td>Average</td><td>25</td><td>Renal, respiratory</td><td>No vaccination</td></tr><tr><td>10</td><td>49</td><td>Winter</td><td>11</td><td>Central</td><td>Average</td><td>14</td><td>Renal, respiratory</td><td>No vaccination</td></tr><tr><td>11</td><td>65</td><td>Summer</td><td>25</td><td>Central</td><td>Poor</td><td>50</td><td>Renal, respiratory, digestive</td><td>d7: Ma5 d14: IB 4/91</td></tr><tr><td>12</td><td>38</td><td>Spring</td><td>7</td><td>Central</td><td>Poor</td><td>26.5</td><td>Renal, respiratory, digestive</td><td>No vaccination</td></tr><tr><td>13</td><td>52</td><td>Autumn</td><td>30</td><td>West</td><td>Poor</td><td>50</td><td>Renal, respiratory, digestive</td><td>d7: H120</td></tr><tr><td>14</td><td>55</td><td>Autumn</td><td>15</td><td>West</td><td>Average</td><td>14.5</td><td>Renal, respiratory</td><td>No vaccination</td></tr></tbody></table>

IBV=Infectious bronchitis virus
the IB 4/91 reference strain (Figure-1). The IBDZ13a strain was characterized as an IB 4/91 variant IBV with 29 nucleic acid substitutions differing from the known IB 4/91 vaccine reference strain sequence, which corresponded to eight amino acid substitutions relative to this reference strain (Table-3). Three of these amino acid substitutions were from a hydrophobic to a hydrophilic amino acid. A change in hydrophilicity altered the epitopes on the S1 protein of the IB DZ13a virus. The predicted epitopes with differences between the field virus and IB 4/91 vaccine strain are shown in Figure-1.

Discussion

Infectious bronchitis has had a devastating effect on broiler farms since the worldwide emergence and increased prevalence of IBV [11,13]. Due to the high rate of mutation and recombination in coronaviruses, new serotypes and genotypes of IBV frequently emerge and have been reported in different regions around the world [11]. More recently, IBV has been associated with nephritis which is now a major problem in broiler flocks worldwide [19-21]. Similar concerns have been reported in Algeria, but no study has been performed so far on IB-associated nephritis [22].

<table><caption>Table-2: IBV-HI antibody titers in broiler flocks suffering from kidney damages.</caption><thead><tr><th rowspan="2">Flocks</th><th colspan="4">Mean titers (log<sub>2</sub>)</th><th colspan="4">Mean titers range (maximum-minimum) (log<sub>2</sub>)</th></tr><tr><th>Qx</th><th>M-41</th><th>D-274</th><th>4/91</th><th>Qx</th><th>M-41</th><th>D-274</th><th>4/91</th></tr></thead><tbody><tr><td>1</td><td>11.00</td><td>5.80</td><td>4.70</td><td>7.00</td><td>11-11</td><td>4-9</td><td>4-9</td><td>6-11</td></tr><tr><td>2</td><td>11.00</td><td>7.00</td><td>4.64</td><td>7.55</td><td>11-11</td><td>5-10</td><td>4-7</td><td>7-9</td></tr><tr><td>3</td><td>9.70</td><td>6.50</td><td>5.20</td><td>7.20</td><td>7-11</td><td>4-11</td><td>4-8</td><td>4-11</td></tr><tr><td>4</td><td>10.82</td><td>6.18</td><td>5.18</td><td>7.00</td><td>9-11</td><td>5-11</td><td>4-9</td><td>4-11</td></tr><tr><td>5</td><td>11.00</td><td>8.09</td><td>6.91</td><td>9.00</td><td>11-11</td><td>6-10</td><td>5-10</td><td>7-11</td></tr><tr><td>6</td><td>11.00</td><td>6.70</td><td>5.10</td><td>6.70</td><td>11-11</td><td>5-8</td><td>4-7</td><td>5-9</td></tr><tr><td>7</td><td>9.78</td><td>9.78</td><td>8.78</td><td>9.89</td><td>7-11</td><td>8-11</td><td>5-11</td><td>8-11</td></tr><tr><td>8</td><td>11.00</td><td>7.75</td><td>5.25</td><td>7.50</td><td>11-11</td><td>7-8</td><td>5-6</td><td>7-8</td></tr><tr><td>9</td><td>10.50</td><td>5.33</td><td>4.50</td><td>5.17</td><td>8-11</td><td>4-9</td><td>4-6</td><td>4-9</td></tr><tr><td>10</td><td>10.14</td><td>4.86</td><td>4.00</td><td>5.00</td><td>5-11</td><td>4-8</td><td>4-4</td><td>4-8</td></tr><tr><td>11</td><td>9.50</td><td>8.33</td><td>5.83</td><td>9.67</td><td>7-11</td><td>6-10</td><td>4-8</td><td>7-11</td></tr><tr><td>12</td><td>8.73</td><td>7.55</td><td>5.91</td><td>8.00</td><td>4-11</td><td>5-9</td><td>4-7</td><td>5-11</td></tr><tr><td>13</td><td>9.80</td><td>8.70</td><td>6.70</td><td>8.80</td><td>7-11</td><td>6-11</td><td>4-11</td><td>8-11</td></tr><tr><td>14</td><td>10.75</td><td>9.38</td><td>6.75</td><td>9.88</td><td>10-11</td><td>8-11</td><td>5-10</td><td>8-11</td></tr></tbody></table>

IBV-HI=Infectious bronchitis virus-hemagglutination inhibition

<table><caption>Table-3: Changes in IBDZ13a amino acid sequence relative to the known IB 4/91 vaccine strain sequence.</caption><thead><tr><th>Position</th><th>Vaccine strain sequence</th><th>Hydrophobicity index</th><th>Sample sequence</th><th>Hydrophobicity index</th><th>hydrophobic/hydrophilic switch</th></tr></thead><tbody><tr><td>253</td><td>Alanine</td><td>1.8</td><td>Threonine</td><td>-0.7</td><td>Yes</td></tr><tr><td>258</td><td>Aspartate</td><td>-3.5</td><td>Glutamate</td><td>-3.5</td><td>No</td></tr><tr><td>261</td><td>Valine</td><td>4.2</td><td>Isoleucine</td><td>4.5</td><td>No</td></tr><tr><td>273</td><td>Glutamate</td><td>-3.5</td><td>Threonine</td><td>-0.7</td><td>No</td></tr><tr><td>286</td><td>Proline</td><td>-1.6</td><td>Serine</td><td>-0.8</td><td>No</td></tr><tr><td>289</td><td>Serine</td><td>-0.8</td><td>Leucine</td><td>3.8</td><td>Yes</td></tr><tr><td>296</td><td>Valine</td><td>4.2</td><td>Glutamine</td><td>-3.5</td><td>Yes</td></tr><tr><td>305</td><td>Serine</td><td>-0.8</td><td>Aspartate</td><td>-3.5</td><td>No</td></tr></tbody></table>

IB=Infectious bronchitis

<figure><img src="image_2.png" /><figcaption>Figure-1: Predicted epitope alignment of deduced amino sequences of HVR S1 gene. New field Algerian isolate and selected infectious bronchitis virus vaccine vial reference strains.</figcaption></figure>
The present study was carried out to investigate
the presence of IBV in broiler flocks and its possible
involvement in kidney damage. We also monitored
the seroprevalence of different IBV serotypes by the
HI test and analyzed the phylogenetic characteristics
of the new IBV strains isolated in Algeria in 2013 by
sequencing. The HI test and virus neutralization test
(VNT) are the most commonly used tests for detect-
ing IBV antibodies [9,23]. Studies show extensive
cross-reactions between the IBV serotypes detected
by the HI test and similar antigenicity detected by the
VNT and HI test [24-26]. However, since VNTs are
not cost-effective for routine examination, HI tests are
commonly used for routine serology [27].

The most frequently detected IBV serotypes in
the broiler flocks with nephritis were IB-QX (100%)
and IB 4/91 (60%), which have been detected for
the 1st time in Algeria by our group. After report-
ing the nephropathogenic form of IB [3], the above
serotypes have been reported in China, Europe, and
Africa and have been seen to be associated with
severe kidney damage, high morbidity, and mortality
in the vaccinated flocks. At present, these serotypes
are the predominant field strains in most poultry
farms worldwide [13,28]. The serological analysis
showed significantly lower titers of IB 4/91 compared
to IB-QX, which could be related to the vaccination
of some flocks with the IB 4/91 strain. De Wit [23]
reported that many factors influence the level of IBV
antibodies, of which the most critical is the presence
of immunity at the time of infection. This is consistent
with the lower anti-IB 4/91 antibody titers seen in the
vaccinated flocks compared to the unvaccinated ones.
Another factor that can reduce the sensitivity of HI
and thus affect the interpretation of the results is the
cross-reaction between the different serotypes, espe-
cially when multiple serotypes of IBV are involved.
We detected cross-reaction between the QX and 4/91
serotypes in many flocks, and 4 flocks showed mixed
infection of three serotypes (QX, 4/91, and M41).
Therefore, the serotype specificity of HI is reduced
after reinfection or vaccination, especially when the
subsequent serotype is heterologous [23].

The S1 protein of IBV determines the genotype,
serotype, and phenotype of IBVs [29]. The S1 gene of
the IBDZ13a field strain was most closely related to
that of the IB 4/91-type strain (Figure-2) as determined
by the criterion of Worthington et al. [13] and Kannan
et al. [30], who classified IBV sequences with <99%
homology to vaccine strains as variants. Our findings
indicate the circulation of virulent strains of IB 4/91,
which have become the predominant genotype in
Africa, Asia, and Europe [28]. However, we did not
see a strong correlation between the HI results which
showed the circulation of the QX and 4/91 serotypes
and RT-PCR which showed the emergence of a new
genotype of the 4/91 strain. This is consistent with the
findings of Kusters et al. [31] who also reported that
RT-PCR and HI results were not exactly correlated,

wherein different serotypes showed considerable dif-
ferences (20-50%) in the amino acid sequences of
the S1 subunit. HI is used for routine testing of sera
from young growing chickens to determine the sero-
type-specific antibody status of a flock [32]. However,
the presence of multiple cross-reactive antibodies
against antigenically unrelated strains, as is often seen
during IB outbreaks, reduces the serodiagnostic value
of HI. Therefore, a combination of serotyping and
genotyping is recommended to identify field strains.
Based on the results of HI and RT-PCR, we conclude
that the IBDZ13a variant of IB4/91 is involved in kid-
ney damage of broiler flocks.

<figure><img src="image_3.png" /><figcaption>**Figure-2:** Neighbor-joining phylogenetic tree with 1000 bootstrap replicates expressing the relations among deduced S1 amino acid sequences of IBDZ13a with selected infectious bronchitis virus reference strains.</figcaption></figure>

When live-attenuated and inactivated vaccines of
IBV were first introduced, they were effective in con-
trolling IB among poultry flocks [33]. However, vac-
cinated flocks in many regions of Algeria have shown
considerable mortality and production losses in recent
years. The isolation of a new IBV strain from the vac-
cinated and unvaccinated flocks can be explained by
two hypotheses. The first hypothesis, based on the
mutations and amino acid alterations in the S1 epi-
tope, is that the IBV strain evolved to escape the host
immune response elicited by the current vaccines [34].
However, this hypothesis is weak since amino acid
alterations do not always translate into differences in
antigenicity or biological function. Furthermore, the
sequence data only provide information regarding the
primary structure of the protein and not the secondary
and tertiary structures which are vital for its biological function and antigenicity [23]. The second hypothesis is the failure to adopt the vaccination programs to the IBV field strains circulating in the farms. In our study, only two flocks received the combined vaccine of IB Ma5 (at day 7) and IB 4/91 (at day 14). The failure to protect broilers against nephritis could also be related to the shorter inter-vaccination interval of 7 days. Terregino et al. [35] demonstrated improved protection against several IB serotypes by vaccinating with two antigenically different IB vaccines given at the interval of 14 days. This approach obviates the need to develop a new IB vaccine for each new emerging serotype. One flock was vaccinated with the Mass vaccine type H120 strain, which, however, did not provide sufficient protection against IBV infection [33,36].

## Conclusion

We identified IBDZ13a, a new nephropathogenic variant of IB 4/91, for the 1st time in Algerian broiler flocks. Its isolation from the vaccinated flocks indicates poor protection afforded by the vaccination programs to the broilers against infection with new IB variants. Further studies are needed to explain the failure of the current vaccines against the new IB4/91 strain and to investigate the cross-protection and pathogenicity of the new genotype.

## Authors' Contributions

AL and MO planned and designed the whole study. AL and HM collected samples, performed technique, and analyzed the data. KO and MO supervised the project and helped during manuscript writing, crosschecking, and revision. All authors read and approved the final manuscript.

## Acknowledgments

The authors gratefully acknowledge the MSD International Laboratory, AMMAN, Jordan, and MSD, Algeria, for providing the facilitation and equipment throughout the study. The authors are also thankful to Entisar and Husam Bakri for technical assistance and help with the HI test analysis.

## Competing Interests

The authors had used the facility of MSD International Laboratory, Jordan but no person from MSD was involved at any stage of the experiment or publication. Also, authors have not received any fund from MSD Algeria.

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