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Zika, yellow fever and dengue viruses are three important flaviviruses that are epidemic and endemic in Central Africa, Middle and South America, Southeast Asia and Pacific Islands. Since these three viral diseases present with similar symptoms and there is a possibility of multiple flaviviruses co-infection, it is important to develop an effective and quick diagnostic tool. Here, we designed and validated a one-step triplex real time reverse transcription polymerase chain reaction (RT-PCR) using TaqMan probes targeting NS5 region for detecting zika virus (ZIKV), dengue virus-4 (DENV-4) and yellow fever virus (YFV). The assay was highly specific for the target viral RNA corresponding to ZIKV, DENV-4 and YFV strains. The sensitivity of the assay was compared to one-step, RT-PCR, it showed a 100-fold higher sensitivity than RT-PCR with ZIKV and DENV and 10-fold higher sensitivity with YFV. In conclusion, the one-step, triplex, TaqMan, real time RT-PCR assay identified and distinguished ZIKV, DENV-4 and YFV by targeting single gene. Key words: Zika, yellow fever, dengue, multiplex real time reverse transcription-polymerase chain reaction (RT-PCR).

The objective of this study was to identify the species and characterize the genetic relationships among mycoplasma isolates from commercial layer hen flocks using 16S-23S rDNA intergenic spacer region (IGSR) sequencing. Twenty-one isolates were obtained from samples collected from commercial layer flocks in four Brazilian states: São Paulo, Minas Gerais, Rio de Janeiro and Espírito Santo. The isolates were recovered from the São Paulo, Rio de Janeiro and Espírito Santo states. Eleven isolates were originated from tracheal swabs, five from shell gland swabs and five from ovary fragment collection. The 16S-23S rDNA IGSR of isolates were amplified by PCR, and the obtained products were subsequently sequenced. The consensus of each isolate was compared to the available sequences using Nucleotide BLAST® to determine the mycoplasma species. A phylogenetic analysis of the Mycoplasma gallisepticum (MG) sequences was performed. Pairwise analyses showed homologies of 99% to 100% with the previously characterized sequences listed in GenBank®. Four Mycoplasma gallinaceum were isolated from three flocks and seven M. pullorum isolates were obtained from a single flock. The other 10 isolates were all identified as MG and were obtained from four flocks. The 16S-23S rDNA IGSR sequencing was a good method to identify Mycoplasma species isolated from field samples, providing fast and reliable results at relatively low costs. The results were also satisfactory for the single-locus sequence typing of MG isolates.

Background: Infectious bursal disease virus (IBDV) is the causative agent of Infectious Bursal Disease (IBD), the disease causes immunosuppression which leads to secondary infections among rearing poultry flocks. Characterization of the virus is important for its control and eradication. The circulating IBDVs are classified on the basis of their antigenic and pathogenic properties. The virus is categorised as classical, variant and very virulent IBDV (vvIBDV). IBDV is a non-envelop, icosahedral double stranded virus. Viral protein 2 (VP2) is the major structural protein of capsid that determines the host-pathogen relationship. The aim of this study was to characterise the IBD virus of Pak-Asian region. Methodology: IBDV suspected flocks were examined in Punjab, Pakistan from 2014–2018. Two hundred and fifty samples were collected with complete history of the disease. The suspected samples were collected from broiler, layer and rural poultry farms. RNA was extracted and hyper-variable region of VP2 gene was amplified using specific primers. Nucleotide sequence of the VP2 gene was determined and its Amino Acid sequence was deduced. Moreover, phylogenetic analysis was also performed. Results: The current classifications based in a hyper-variable region of the capsid protein VP2 (hvVP2), classification of IBDVs is split into newly proposed geno-groups according to Jackwood group. Among these prevailing, some IBDVs are limited geographically whereas, others are reported cosmopolitan. Genetic alterations are continuously playing role in evolution of new strains of the virus. Conclusion: During this study it was found that isolates of IBDV fall in first three geno-groups. Most of the geno-groups are prevalent around the world, whereas the mutated and re-assorted ones are confined in particular areas of the globe. Keywords: IBDV, Classification, Geno-groups, Pak-Asian region, Phylogenetic analysis

Fowl adenovirus-4 is an infectious agent causing Hydropericardium syndrome in chickens. Adenovirus are non-enveloped virions having linear, double stranded DNA. Viral genome codes for few structural and non structural proteins. 100K is an important non-structural viral protein. Open reading frame for coding sequence of 100K protein was cloned with oligo histidine tag and expressed in Escherichia coli as a fusion protein. Nucleotide sequence of the gene revealed that 100K gene of FAdV-4 has high homology (98%) with the respective gene of FAdV-10. Recombinant 100K protein was expressed in E. coli and purified by nickel affinity chromatography. Immunization of chickens with recombinant 100K protein elicited significant serum antibody titers. However challenge protection test revealed that 100K protein conferred little protection (40%) to the immunized chicken against pathogenic viral challenge. So it was concluded that 100K gene has 2397 bp length and recombinant 100K protein has molecular weight of 95 kDa. It was also found that the recombinant protein has little capacity to affect the immune response because in-spite of having an important role in intracellular transport & folding of viral capsid proteins during viral replication, it is not exposed on the surface of the virus at any stage.

Salmonella enterica serovar Enteritidis is a major foodborne pathogen in the United States, causing gastroenteritis in humans, primarily through consumption of contaminated eggs. Chickens are the reservoir host of S . Enteritidis. In layer hens, S . Enteritidis colonizes the intestine and migrates to various organs, including the oviduct, leading to egg contamination. This study investigated the efficacy of in-feed supplementation with trans -cinnamaldehyde (TC), a generally recognized as safe (GRAS) plant compound obtained from cinnamon, in reducing S . Enteritidis cecal colonization and systemic spread in layers. Additionally, the effect of TC on S . Enteritidis virulence factors critical for macrophage survival and oviduct colonization was investigated in vitro . The consumer acceptability of eggs was also determined by a triangle test. Supplementation of TC in feed for 66 days at 1 or 1.5% (vol/wt) for 40- or 25-week-old layer chickens decreased the amounts of S . Enteritidis on eggshell and in yolk ( P < 0.001). Additionally, S . Enteritidis persistence in the cecum, liver, and oviduct in TC-supplemented birds was decreased compared to that in controls ( P < 0.001). No significant differences in feed intake, body weight, or egg production in birds or in consumer acceptability of eggs were observed ( P > 0.05). In vitro cell culture assays revealed that TC reduced S . Enteritidis adhesion to and invasion of primary chicken oviduct epithelial cells and reduced S . Enteritidis survival in chicken macrophages ( P < 0.001). Follow-up gene expression analysis using real-time quantitative PCR (qPCR) showed that TC downregulated the expression of S . Enteritidis virulence genes critical for chicken oviduct colonization ( P < 0.001). The results suggest that TC may potentially be used as a feed additive to reduce egg-borne transmission of S . Enteritidis.

We used a platform of self-assembling protein nanoparticles (SAPNs) to develop a self-adjuvanted vaccine for avian influenza virus (AIV). A SAPNs named SA-MC-Penn was designed to display the ectodomain of influenza matrix protein 2 (M2e) as a tetramer and Helix C (Hel C) of hemagglutinin as a trimer. Flagellin domainsexpressed within a peptide chain were co-assembled into the SAPN core serving as a built-in adjuvant. SA-MC-Penn was administered every two weeks for three times via the intramuscular (IM) or eye drop (ED) routes in chickens starting at four-weeks of age. Sera were collected from immunized chickens at twelve-weeks of age prior to challenge with a highly pathogenic (HP) H5N2 strain of AIV. ELISA results demonstrated that high titers of serum antibodies were induced in the IM immunized SAPNs. Upon challenge with HP AIV, 63 % and 30% of chickens the SAPNs IM and SAPN ED groups, respectively, survived. A reduction in challenge virus shedding from oropharyngeal swabbings of the SAPNs IM vaccinates was observed on days two and 14-post challenge. These results show the SAPNs vaccine prototype was partially protective against HPAIV.

Avian Colibacillosis is among the major causes of economic loss in the poultry industry worldwide, with a more vivid impact on developing countries. The involvement of several bacteria has made it challenging to develop effective vaccines for this disease, particularly because it is notoriously difficult to make a vaccine that contains all the contributing pathogenic bacteria. Here, we report the design and fabrication of a bacterial ghost (BG) of E. coli O78:K80, which is among the major bacterial serotypes responsible for this disease. The generated ghost is then exploited as a homologous vaccine against Avian Colibacillosis. We demonstrate that hole formation in the cell wall of E. coli O78:K80 can happen properly in optical densities as high as 0.8 compared to the 0.3–0.4 standard for bacteria like E. coli TOP10. This is especially advantageous for mass production of this ghost which is a vital factor in development of any BG-based vaccine. Compared to E. coli TOP10, we faced a great challenge in transforming the wild type bacteria with the E-lysis plasmid which was probably due to higher thickness of the cell wall in O78:K80. This, however, was addressed by treating the cell wall with a different combination of ions.The vaccine was administered to Ross 308 broiler chickens via injection as well as through their respiratory system at a dose of 1010 BGs, repeated 3 times at weekly intervals. Chickens were then challenged with the wild type O78:K80 at a dose of 1011 bacteria together with Infectious Bronchitis H120 vaccine (as immunosuppressant) one week after the last immunization. Air sac lesions were significantly reduced in BG vaccinated groups in comparison with the control group. The levels of IFNγ, IgA and IgY were measured in the serum of immunized chickens as an indication of immune response and were compared with those of the chickens vaccinated with killed bacteria. The results show that O78:K80 BG can be used as an efficient homologous vaccine against Colibacillosis disease in poultry. We expect our findings can serve as the starting point for designing more sophisticated vaccines that contain all three major pathogenic bacteria involved in avian Colibacillosis.

The objective of this study was to evaluate the susceptibility of metallo-β-lactamase (MBL)-producing Acinetobacter baumannii (A. baumannii) clinical isolates to biocides. We also determined the prevalence and correlation of efflux pump genes, class 1 integron and MBL encoding genes. In addition, blaVIM, blaNDM-1, qacE and qacEΔ1 nucleotide sequence analysis was performed and compared to sequences retrieved from GenBank at the National Center for Biotechnology Information database. A. baumannii had a resistance rate to carbapenem of 71.4% and 39.3% and was found to be a MBL producer. The minimum inhibitory concentrations (MICs) of chlorhexidine and cetrimide were higher than the recommended concentrations for disinfection in 54.5% and 77.3% of MBL-positive isolates respectively and their MICs were significantly higher among qac gene-positive isolates. Coexistence of qac genes was detected in 68.1% and 50% of the isolates with blaVIM and blaNDM-1 respectively. There was a significant correlation between the presence of qac genes and MBL-encoding blaVIM and blaNDM-1 genes. Each of the blaNDM-1, blaVIM, qacE and qacEΔ1 DNA sequences showed homology with each other and with similar sequences reported from other countries. The high incidence of Verona integron-encoded metallo-β-lactamases (VIM) and New-Delhi-metallo-β-lactamase (NDM) and qac genes in A. baumannii highlights emerging therapeutic challenges for being readily transferable between clinically relevant bacteria. In addition reduced susceptibility to chlorhexidine and cetrimide and the potential for cross resistance to some antibiotics necessitates the urgent need for healthcare facilities to periodically evaluate biocides efficacy, to address the issue of antiseptic resistance and to initiate a “biocidal stewardship”.

The objective of this study was to evaluate the susceptibility of metallo-β-lactamase (MBL)-producing Acinetobacter baumannii (A. baumannii) clinical isolates to biocides. We also determined the prevalence and correlation of efflux pump genes, class 1 integron and MBL encoding genes. In addition, blaVIM, blaNDM-1, qacE and qacEΔ1 nucleotide sequence analysis was performed and compared to sequences retrieved from GenBank at the National Center for Biotechnology Information database. A. baumannii had a resistance rate to carbapenem of 71.4% and 39.3% and was found to be a MBL producer. The minimum inhibitory concentrations (MICs) of chlorhexidine and cetrimide were higher than the recommended concentrations for disinfection in 54.5% and 77.3% of MBL-positive isolates respectively and their MICs were significantly higher among qac gene-positive isolates. Coexistence of qac genes was detected in 68.1% and 50% of the isolates with blaVIM and blaNDM-1 respectively. There was a significant correlation between the presence of qac genes and MBL-encoding blaVIM and blaNDM-1 genes. Each of the blaNDM-1, blaVIM, qacE and qacEΔ1 DNA sequences showed homology with each other and with similar sequences reported from other countries. The high incidence of Verona integron-encoded metallo-β-lactamases (VIM) and New-Delhi-metallo-β-lactamase (NDM) and qac genes in A. baumannii highlights emerging therapeutic challenges for being readily transferable between clinically relevant bacteria. In addition reduced susceptibility to chlorhexidine and cetrimide and the potential for cross resistance to some antibiotics necessitates the urgent need for healthcare facilities to periodically evaluate biocides efficacy, to address the issue of antiseptic resistance and to initiate a “biocidal stewardship”.

Most of the killed, inactivated, or live-attenuated pathogen vaccines are now replaced by modern vaccines containing isolated, highly purified antigenic protein subunits which are safer than live viruses or deactivated viruses. Another strategy is the development of nanoparticles which can mimic the repetitiveness, geometry, size and shape of the host-pathogen surface and provide improved stability and long lasting immunogenicity, as well as serve as vehicles to deliver multiple copies of the antigens to the target cells. Several interesting advances have been made recently in the area of protein nanotechnology and here, we provide a concise review of one such novel nanoparticle called FluoDot which may be effectively used as a delivery system in some veterinary medicine applications.

Avian reovirus infection causes considerable economic loss to the commercial poultry industry. Live-attenuated vaccine strain S1133 (v-S1133, derived from parent strain S1133) is considered the safest and most effective vaccine and is currently used worldwide. To identify the genes responsible for its attenuation, DNA sequences of open reading frames (ORF) of S1133 and its parent strains S1133, 1733, 526, and C78 along with three field isolates (GuangxiR1, GuangxiR2, and GX110058) and one isolate (GX110116) from a vaccinated chicken were performed. The sequence data were compared with available sequences in nucleotide sequence databases of American (AVS-B, 138, 176) and Chinese (C-98 and T-98) origin. Sequence analysis identified that several v-S1133 specific nucleotide substitutions existed in the ORFs of λA, λB, λC, μA, μB, μNS, σA, σB, and σNS genes. The v-S1133 strain could be differentiated from the field-isolated strains based on single nucleotide polymorphisms. Phylogenetic analysis revealed that v-S1133 shared the highest sequence homologies with S1133 and reovirus isolates from China, grouped together in one cluster. Chinese isolates were clearly more distinct from the American reovirus AVS-B strain, which is associated with runting-stunting syndrome in broilers.

The poultry industry has emerged as one of the largest and fastest growing public sectors in the developed and developing countries. Unfortunately, this industry is under a major threat from diseases that are viral (Newcastle disease, infectious bursal disease, influenza, hydropericardium syndrome), bacterial (colibacillosis, pasteurellosis, salmonellosis, mycoplasmosis), parasitic (coccidiosis, histoplasmosis) or nutritional (dyschondroplasia, osteoporosis). Among these diseases, hydropericardium syndrome (HPS) is one of the important emerging diseases occurring in the specific areas of the world where broilers (chickens) are reared under intensive conditions. HPS was first observed in 1987 at Angara Goth, an area near Karachi, Pakistan, where broilers are raised. Since then, HPS has been reported in many countries of the world. From these reported cases, an adenovirus that was either isolated from or visualized electron microscopically in the liver of affected broilers has been implicated in the syndrome. The syndrome has been reproduced by inoculation of isolated fowl adenovirus (FAdV) strains, and hence, the syndrome is also called infectious hydropericardium syndrome. To our knowledge, HPS has not been observed in humans, so it is not considered a zoonotic disease, but it is of economic importance and causes huge losses to the poultry industry. Efforts have been made to develop conventional vaccines against this disease, which were formulated from infected liver homogenate. Formalin-inactivated liver organ vaccines have failed to protect the poultry industry. Hence, there is a dire need to develop a suitable vaccine to combat this disease. Currently, recombinant vaccine candidates are being developed by using molecular biology and biotechnological approaches for the prevention and control of infectious diseases, including HPS. Therefore, it is suggested that the immunogenicity of these recombinant proteins should be evaluated for their use as subunit vaccines.

Current influenza vaccines should be improved by the addition of universal influenza vaccine antigens in order to protect against multiple virus strains. We used our self-assembling protein nanoparticles (SAPNs) to display the two conserved influenza antigens M2e and Helix C in their native oligomerization states. To further improve the immunogenicity of the SAPNs, we designed and incorporated the TLR5 agonist flagellin into the SAPNs to generate self-adjuvanted SAPNs. We demonstrate that addition of flagellin does not affect the ability of SAPNs to self-assemble and that they are able to stimulate TLR5 in a dose-dependent manner. Chickens vaccinated with the self-adjuvanted SAPNs induce significantly higher levels of antibodies than those with unadjuvanted SAPNs and show higher cross-neutralizing activity compared to a commercial inactivated virus vaccine. Upon immunization with self-adjuvanted SAPNs, mice were completely protected against a lethal challenge. Thus, we have generated a self-adjuvanted SAPN with a great potential as a universal influenza vaccine.

The efficacies of trans -cinnamaldehyde (TC) and eugenol (EG) for reducing Salmonella enterica serovar Enteritidis colonization in broiler chickens were investigated. In three experiments for each compound, 1-day-old chicks ( n = 75/experiment) were randomly assigned to five treatment groups ( n = 15/treatment group): negative control (-ve S . Enteritidis, -ve TC, or EG), compound control (-ve S . Enteritidis, +ve 0.75% [vol/wt] TC or 1% [vol/wt] EG), positive control (+ve S . Enteritidis, -ve TC, or EG), low-dose treatment (+ve S . Enteritidis, +ve 0.5% TC, or 0.75% EG), and high-dose treatment (+ve S . Enteritidis, +ve 0.75% TC, or 1% EG). On day 0, birds were tested for the presence of any inherent Salmonella ( n = 5/experiment). On day 8, birds were inoculated with ∼8.0 log 10 CFU S . Enteritidis, and cecal colonization by S . Enteritidis was ascertained ( n = 10 chicks/experiment) after 24 h (day 9). Six birds from each treatment group were euthanized on days 7 and 10 after inoculation, and cecal S . Enteritidis numbers were determined. TC at 0.5 or 0.75% and EG at 0.75 or 1% consistently reduced ( P < 0.05) S . Enteritidis in the cecum (≥3 log 10 CFU/g) after 10 days of infection in all experiments. Feed intake and body weight were not different for TC treatments ( P > 0.05); however, EG supplementation led to significantly lower ( P < 0.05) body weights. Follow-up in vitro experiments revealed that the subinhibitory concentrations (SICs, the concentrations that did not inhibit Salmonella growth) of TC and EG reduced the motility and invasive abilities of S . Enteritidis and downregulated expression of the motility genes flhC and motA and invasion genes hilA , hilD , and invF . The results suggest that supplementation with TC and EG through feed can reduce S . Enteritidis colonization in chickens.

Two nonstructural genes, sigmaNS and P17, of avian reovirus (ARV) were cloned into the expression plasmid vector PGEX4T-1. Expressed proteins for sigmaNS and P17 of avian reovirus were purified and used as antigens. Three indirect sigmaNS enzyme-linked immunosorbent assays (ELISAs), sigmaNS-ELISA, P17-ELISA and sigmaNS-P17-ELISA were optimized and used as specific tests. Serum samples from reovirus-infected and vaccinated SPF chickens were tested with the three ELISAs and an agar gel precipitin (AGP) method. ELISAs specific for sigmaNS, P17 and sigmaNS-P17 were able to detect specific antibodies for avian reovirus in 88.9%, 61.1%, and 88.9% in infected samples, respectively, whereas the AGP detected 55.6% of the infected samples. The detection rates of ELISA specific antibodies for sigmaNS, P17 and sigmaNS-P17 on sera of vaccinated chickens were 6.7%, 0% and 6.7%. However, in comparison the AGP method detected 60.6% of antibodies in serum samples from vaccinated chickens. The results showed that the use of ELISAs specific for the nonstructural proteins might be able to distinguish between reovirus vaccinated and infected chickens. Further studies are in progress to validate these recombinant protein-based ELISAs under field conditions.

Infectious Bronchitis Virus (IBV) can cause high mortality, poor weight gain and low feed efficiency of infected chickens, which leading to considerable economic losses to the poultry industry. In this study, a plasmid DNA pTracer-CVM2-IBVS (abbr: pCMV-S) that expresses the immunogenic S glycoprotein genes of IBV serotype Massachusetts 41 was inoculated in ovo into 18-days-old embryonating Specific-PathogenFree (SPF) chicken eggs as a vaccine. Recombinant chicken interferon type I alpha (rChIFN-") was added as an adjuvant to enhance the immunogenicity of the vaccine. After hatch, birds were challenged at 4 weeks of age with a homologous pathogenic Massachusetts 41 field type IBV. Birds receiving a single dose of pCMV-S vaccine in ovo showed mild clinical signs and were protected at the level of 66%, whereas groups receiving a combination of the pCMV-S with 2000 IU or 500 IU of rChIFN-" showed protection at the level of 83 and 89% respectively. The pCMV-S + 500 IU rChIFN-" vaccinated group, when boosted with a live attenuated commercial IBV vaccine at 2 weeks of age indicated a significant protection (> 92 %) against IBV challenge. At 5 days post challenge, no clinical signs were seen from the birds vaccinated with 5 ug of pCMVS +500 IU rChIFN and the birds received booster with commercial IBV vaccine. RT-PCR based diagnostic test indicated detecting IBV infection was reduced in birds received pCMV-S with 2000 IU or 500 IU of rCHIFN-". This study indicates that the application of rChIFN-" as an adjuvant with pTracer-CMV-IBVS vaccine expressing S glycoprotein gene was somewhat effective in controlling IBV infection. Additionally an increase in protection against the homologous IBV challenge was observed in birds boosted with the live attenuated IBV commercial vaccine.

Salmonella Enteritidis is a major foodborne pathogen for which chickens serve as reservoir hosts. Reducing Salmonella Enteritidis carriage in chickens would reduce contamination of poultry meat and eggs with this pathogen. We investigated the prophylactic efficacy of feed supplemented with caprylic acid (CA), a natural, generally recognized as safe eight-carbon fatty acid, for reducing Salmonella Enteritidis colonization in chicks. One hundred commercial day-old chicks were randomly divided into five groups of 20 birds each: CA control (no Salmonella Enteritidis, CA), positive control (Salmonella Enteritidis, no CA), negative control (no Salmonella Enteritidis, no CA), and 0.7 or 1% CA. Water and feed were provided ad libitum. On day 8, birds were inoculated with 5.0 log CFU of Salmonella Enteritidis by crop gavage. Six birds from each group were euthanized on days 1, 7, and 10 after challenge, and Salmonella Enteritidis populations in the cecum, small intestine, cloaca, crop, liver, and spleen were enumerated. The study was replicated three times. CA supplementation at 0.7 and 1% consistently decreased Salmonella Enteritidis populations recovered from the treated birds. Salmonella Enteritidis counts in the tissue samples of CA-treated chicks were significantly lower (P < 0.05) than those of control birds on days 7 and 10 after challenge. Feed intake and body weight did not differ between the groups. Histological examination revealed no pathological changes in the cecum and liver of CA-supplemented birds. The results suggest that prophylactic CA supplementation through feed can reduce Salmonella Enteritidis colonization in day-old chicks and may be a useful treatment for reducing Salmonella Enteritidis carriage in chickens.

Three isolates of H9N2 Avian Influenza viruses (AIV) were isolated from chickens in Guangxi province. Eight pairs of specific primers were designed and synthesized according to the sequences of H9N2 at GenBank. phylogenetic analysis showed a high degree of homology between the Guangxi isolates and isolates from Guangdong and Jiangsu provinces, suggesting that the Guangxi isolates originated from the same source. However, the eight genes of the three isolates from Guangxi were not in the same sublineages in their respective phylogenetic trees, which suggests that they were products of natural reassortment between H9N2 avian influenza viruses from different sublineages. The 9 nucleotides ACAGAGATA which encode amino acids T, G, I were absent between nucleotide 205 and 214 in the open reading frame of the NA gene in the Guangxi isolates. AIV strains that infect human have, in their HA proteins, leucine at position 226. The analysis of deduced amino acid sequence of HA proteins showed that position 226 of these isolates contained glycine instead of leucine, suggesting that these three isolates differ from H9N2 AIV strains isolated from human infections.

Three isolates of H9N2 subtype of influenza virus have been isolated from chickens in Guangxi province. In this study, eight full-length genes of each of the H9N2 isolates, A/Chicken/Guangxi/1/00, A/Chicken/Guangxi/14/00, and A/Chicken/Guangxi/17/00, were obtained. Sequence analysis and phylogenetic studies were conducted by comparing eight genes of each isolate with those of the available H9N2 strains at GenBank. Results showed a high degree of homology between the Guangxi isolates and isolates from Guangdong and Jiangsu provinces, suggesting that Guangxi isolates originated from the same source. However, the eight genes of these three isolates from Guangxi were not in the same sublineages in the phylogenic trees, which suggest that they were products of natural reassortment between H9N2 avian influenza viruses from different sublineages. The 9 nucleotides ACAGAGATA encoding amino acids T, E, I were absent between nucleotide 205 and 214 in the open reading frame of NA genes in the Guangxi isolates. AIV strains that infect human have in their HA proteins, Leucine at position 226. The analysis of deduced amino acid sequence of HA proteins of C/GX/1/00, C/GX/14/00, and C/GX/17/00 showed that the position 226 of these isolates was glycine instead of leucine, suggesting that these three isolates differ from H9N2 AIV strains isolated from human infections.

β-Lactams disrupt bacterial cell wall synthesis, and these agents are the most widely used antibiotics. One of the principle mechanisms by which bacteria resist the action of β-lactams is by producing β-lactamases, enzymes that degrade β-lactams. In Gram-negative bacteria, production of β-lactamases is often induced in response to the antibiotic-associated damage to the cell wall. Here, we have identified a previously unidentified mechanism that governs β-lactamase production. In the Gram-negative enteric pathogen Vibrio parahaemolyticus, we found a histidine kinase/response regulator pair (VbrK/VbrR) that controls expression of a β-lactamase. Mutants lacking either VbrK or VbrR do not produce the β-lactamase and are no longer resistant to β-lactam antibiotics. Notably, VbrK autophosphorylation is activated by β-lactam antibiotics, but not by other lactams. However, single amino acid substitutions in the putative periplasmic binding pocket of VbrK leads its phosphorylation in response to both β-lactam and other lactams, suggesting that this kinase is a β-lactam receptor that can directly detect β-lactam antibiotics instead of detecting the damage to cell wall resulting from β-lactams. In strong support of this idea, we found that purified periplasmic sensor domain of VbrK binds penicillin, and that such binding is critical for VbrK autophosphorylation and β-lactamase production. Direct recognition of β-lactam antibiotics by a histidine kinase receptor may represent an evolutionarily favorable mechanism to defend against β-lactam antibiotics.

Salmonella Enteritidis (SE) is a major foodborne pathogen responsible for causing gastrointestinal infections in humans, predominantly due to the consumption of contaminated eggs. In layer hens, SE colonizes the intestine and migrates to various organs, including the oviduct, thereby leading to egg yolk and shell contamination. This study investigated the efficacy of caprylic acid (CA), a medium-chain fatty acid, in reducing SE colonization and egg contamination in layers. Caprylic acid was supplemented in the feed at 0%, 0.7%, or 1% (vol/wt) from day 1 of the experiment. Birds were challenged with 10(10) log colony-forming units (CFU)/mL of SE by crop gavage on day 10, and re-inoculated (10(10) log CFU/mL) on day 35. After 7 days post first inoculation, eggs were collected daily and tested for SE on the shell and in the yolk separately. The birds were sacrificed on day 66 to determine SE colonization in the ceca, liver, and oviduct. The consumer acceptability of eggs was also determined by triangle test. The experiment was replicated twice. In-feed supplementation of CA (0.7% and 1%) to birds consistently decreased SE on eggshell and in the yolk (p0.05). Supplementation of CA at 1.0% decreased SE population to ≈14% on the shell and ≈10% in yolk, when compared to control birds, which yielded ≈60% positive samples on shell and ≈43% in yolk. Additionally, SE populations in the cecum and liver were reduced in treated birds compared to control (p0.05). No significant difference in egg production, body weight, or sensory properties of eggs was observed (p0.05). The results suggest that CA could potentially be used as a feed additive to reduce eggborne transmission of SE.

Thirty-one Newcastle disease viruses (NDVs) isolated from domestic and wild birds in Shandong Province, China (2006-2014) were characterized genetically, pathogenically and antigenically. Phylogenetic analysis classified the viruses into a single genotype under Class I, and four genotypes under Class II. The nineteen viruses classified in genotype VII of Class II were velogenic, while the other viruses were either mesogenic or lentogenic to chickens. Some NDV isolates (17/23) showed no neutralizing reactivity with a monoclonal antibody developed against the HN protein of the LaSota strain, reflecting the mutation at the related antigenic epitope. When challenged with two genotype VII NDV isolates, LaSota-vaccinated SPF chickens were prevented from disease development, but virus shedding was detected for at least 5 days post challenge. Circulation of the same NDV isolate for up to 13 years suggested the role of an environmental reservoir in NDV perpetuation, and reinforced the importance of strict biosecurity measures in addition to vaccination for disease control.

A reverse transcriptase-polymerase chain reaction (RT-PCR) was developed and optimized for the detection of avian encephalomyelitis virus (AEV). A pair of primers was prepared based on the VP2 gene of the structural protein P1 region of the AEV genome. An avian encephalomyelitis virus-specific 619-base pair cDNA product was amplified by these primers from five reference/field strains of AEVs but not from 10 other avian pathogenic viruses and bacteria. The RT-PCR assay developed in this study was found to be sensitive and specific with as little as 10 pg of avian encephalomyelitis virus RNA detected using gel electrophoresis. Furthermore, AEV-RT-PCR was able to detect AE virus from chicken embryo brain at 3 days postinoculation as compared with the AE agar gel precipitation test (AGP), which required up to 11 days of incubation in the embryos.

Aims: To evaluate the ability of Salmonella enterica ser. Enteritidis outer membrane proteins (OMPs) of 75·6 and 82·3 kDa to inhibit or reduce in vivo colonization of S. Enteritidis on intestinal mucosa in chickens. Methods and Results: Nine-week-old specific-pathogen-free chickens were subcutaneously immunized with 75·6 or 82·3 kDa protein, and challenged with a virulent strain of S. Enteritidis. Chickens were killed, and portions of small intestine and caecum were removed at necropsy. The population of S. Enteritidis attached to chicken intestinal mucosa was determined. The population of S. Enteritidis recovered from the small intestine and caecum of chickens immunized with 75·6 or 82·3 kDa protein was significantly (P

A new high-throughput GenomeLab Gene Expression Profiler (GeXP) analyser-based multiplex PCR assay was developed for the detection of eight reproductive and respiratory pathogens in swine. The reproductive and respiratory pathogens include North American porcine reproductive and respiratory syndrome virus (PRRSV-NA), classical swine fever virus (CSFV), porcine circovirus 2 (PCV-2), swine influenza virus (SIV) (including H1 and H3 subtypes), porcine parvovirus (PPV), pseudorabies virus (PRV) and Japanese encephalitis virus (JEV). Nine pairs of specific chimeric primers were designed and used to initiate PCRs, and one pair of universal primers was used for subsequent PCR cycles. The specificity of the GeXP assay was examined using positive controls for each virus. The sensitivity was evaluated using serial ten-fold dilutions of in vitro-transcribed RNA from all of the RNA viruses and plasmids from DNA viruses. The GeXP assay was further evaluated using 114 clinical specimens and was compared with real-time PCR/single RT-PCR methods. The specificity of the GeXP assay for each pathogen was examined using single cDNA/DNA template. Specific amplification peaks of the reproductive and respiratory pathogens were observed on the GeXP analyser. The minimum copies per reaction detected for each virus by the GeXP assay were as follows: 1000 copies/μl for PRV; 100 copies/μl for CSFV, JEV, PCV-2 and PPV; and 10 copies/μl for SIV-H1, SIV-H3 and PRRSV-NA. Analysis of 114 clinical samples using the GeXP assay demonstrated that the GeXP assay had comparable detection to real-time PCR/single RT-PCR. This study demonstrated that the GeXP assay is a new method with high sensitivity and specificity for the identification of these swine reproductive and respiratory pathogens. The GeXP assay may be adopted for molecular epidemiological surveys of these reproductive and respiratory pathogens in swine populations.

The spike 1 (S1) surface glycoprotein of infectious bronchitis virus (IBV) is the major inducer of the generation of virus neutralizing antibodies, and the administration of purified S1 has been shown to elicit a protective immune response against virulent virus challenge. On the basis of these observations, recombinant fowl poxvirus (rFPV) containing a cDNA copy of the S1 gene of IBV Mass 41 (rFPV-S1) was constructed and its immunogenicity and vaccine potential were evaluated. Initially, rFPV-S1 was shown to express the S1 in vito by indirect immunofluorescence staining and western blot analyses. Later, in vivo expression was demonstrated by the detection of IBV-specific serum immunoglobulin G and neutralization antibodies in the sera of chickens immunized with rFPV-S1. That the recombinant virus elicited anti-IBV protective immunity was indicated by the manifested, relatively mild clinical signs of disease, decreased titers of recovered challenge virus, and less severe histologic changes of the tracheas in virulent IBV Mass 41-challenged chickens previously receiving rFPV-S1 as compared with parental fowl poxvirus (FPV)-vaccinated control birds. In contrast, chickens immunized with either recombinant or parental FPV were resistant to a subsequent virulent FPV challenge. As to a preferred method of immunization, wing web administration appeared to be superior to the subcutaneous route because a greater percentage of birds vaccinated by the former protocol exhibited an anti-IBV humoral immune response. Thus, rFPV-S1 has potential as a poultry vaccine against both fowl pox and infectious bronchitis.

We report here the full-length RNA genomic sequence of the bovine viral diarrhea virus (BVDV) strain GX4, isolated from a cow in southern China. Studies indicate that BVDV GX4 belongs to the BVDV-1b subtype. This report will help in understanding the epidemiology and molecular characteristics of BVDV in southern China cattle.

Equine Lyme disease is difficult to diagnose because of its nonspecific clinical signs and the high incidence of subclinical infection in endemic regions. In this study we compared serology, antigen presence, hematology, blood chemistries and clinical presentation of 22 horses from a highly endemic region that were clinically diagnosed with Lyme disease to that of 21 clinically normal horses from the same region. We found that horses clinically diagnosed with Lyme disease were more likely to have Borrelia burgdorferi spirochetal DNA in their blood and urine, have a higher percentage of positive immunoblots containing antibodies to certain B. burgdorferi proteins, and tend to have higher ELISA titers than healthy horses from the same region. These results may help to improve diagnostic testing for equine Lyme disease.

Infectious bronchitis virus (IBV), the prototype of the coronavirus family, is an enveloped, single-stranded RNA virus with a genome size of approximately 27.6 kilobase. Infectious bronchitis virus causes an acute, highly contagious respiratory and urogenital disease of chickens which results in significant economic losses in commercial broilers, layers and breeders. A rapid, highly sensitive and specific method is needed in the differential diagnosis of infections of different serotypes. A multiplex polymerase chain reaction (PCR) method was developed and optimized to simultaneously detect Massachusetts (Mass) and Arkansas (Ark) serotypes of IBV. One common primer and two serotype specific primers were chosen from the S1 gene sequences of IBV and used in one PCR reaction. Under optimized PCR conditions, two serotype specific PCR products, 1026 bp for Mass and 896 bp for Ark, respectively, were amplified and detected by agarose gel electrophoreses. The specificity of the technique was verified by using 20 different strains and isolates of IBV, and other avian bacterial and viral pathogens. Using a serial 10-fold dilution of the artificial mixture of both Mass and Ark samples, the detection limit was found to be 5 pg RNA after 35 cycles of PCR. The multiplex PCR was able to detect and differentiate both serotypes in embryonated eggs that were co-infected with different EID50virus titers of Mass 41 and Ark 99. The multiplex PCR developed in this study will be valuable for rapid identification, differential diagnosis, and epidemiological studies of these two serotypes of IBV infections.

Diagnosticiansshould be aware of the possibilities of these concurrent viralinfections in raccoons with neurologic disorders.Acknowledgements We thank G. Moser, B. Lehman, andS. Hindman for technical assistance. This work was sup-ported by grants from the Pennsylvania Department of Ag-riculture and the Laboratory of Large Animal Pathology,University of Pennsylvania.References

A new, rapid, and high-throughput GenomeLab Gene Expression Profiler (GeXP) analyzer-based multiplex PCR method was developed for simultaneous detection and differentiation of nine avian respiratory pathogens. The respiratory pathogens included in this study were avian influenza subtypes H5, H7, and H9, infectious bronchitis virus (IBV), Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS) and Haemophilus paragallinarum (HPG). Ten pairs of primers were designed using conserved and specific sequence genes of AIV subtypes and respiratory pathogens from GenBank. Single and mixed pathogen cDNA/DNA templates were used to evaluate the specificity of the GeXP-multiplex assay. The corresponding specific DNA products were amplified for each pathogen. The specific DNA product amplification peaks of nine respiratory pathogens were observed on the GeXP analyzer. Non-respiratory avian pathogens, including chicken infectious anemia virus, fowl adenovirus, avian reovirus and infectious bursal disease virus, did not produce DNA products. The detection limit for the GeXP-multiplex assay was determined to be 100 copies/μl using various pre-mixed plasmids/ssRNAs containing known target genes of the respiratory pathogens. Further, GeXP-multiplex PCR assay was 100% specific when 24 clinical samples with respiratory infections were tested in comparison with conventional PCR method. The GeXP-multiplex PCR assay provides a novel tool for simultaneous detection and differentiation of nine avian respiratory pathogens.

The growth of spoilage bacteria results in a shorter shelf life of meat, causing economic losses to the meat industry. Based on 23S rDNA sequence data of Pseudomonas aeruginosa , two primers designated as PF (23 bases) and PR (20 bases) were synthesized for use in the polymerase chain reaction. A unique 207-base-pair DNA product from nine different bacteria typically associated with meat spoilage was amplified by the primers. Dot blot analysis with the internal DNA probe specific for the amplified products confirmed that the amplified DNA sequence is specific for the spoilage bacteria studied.

A duplex quantitative real-time polymerase chain reaction (dq-PCR) assay was optimized to simultaneously detect Haplosporidium spp. and Perkinsus spp. of shellfish in one reaction. Two sets of specific oligonucleotide primers for Haplosporidium spp. and Perkinsus spp., along with two hydrolysis probes specific for each parasite group, were used in the assay. The dq-PCR results were detected and analyzed using the Light Cycler 2.0 software system. The dq-PCR identified and differentiated the two protozoan parasite groups. The sensitivity of the dq-PCR assay was 200 template copies for both Haplosporidium spp. and Perkinsus spp. No DNA product was amplified when known DNA from Marteilia refringens, Toxoplasma gondii, Bonamia ostreae, Escherichia coli, Cymndinium spp., Mykrocytos mackini, Vibrio parahaemolyticus, and shellfish tissue were used as templates. A total of 840 oyster samples from commercial cultivated shellfish farms from two coastal areas in China were randomly collected and tested by dq-PCR. The detection rate of Haplosporidium spp. was 8.6 % in the Qindao, Shandong coastal area, whereas Perkinsus spp. was 8.3 % coastal oysters cultivated from shellfish farms of Beihai, Guangxi. The dqPCR results suggested that Haplosporidium spp. was prevalent in oysters from Qindao, Shandong, while Perkinsus spp. was prevalent in oysters from the coastal areas of Beihai, Guangxi. This dq-PCR could be used as a diagnostic tool to detect Haplosporidium spp. and Perkinsus spp. in cultivated shellfish.

Hydropericardium syndrome (HPS) is a disease of poultry that is caused by fowl adenovirus-4. Inactivated liver homogenate from diseased birds is still the choice of vaccine in some countries which disseminates numerous pathogens along with inactivated virus. Moreover incomplete attenuation or inactivation, reversion to virulence and the oncogenic potential/genetic instability of the adenoviruses have prevented their use in routine vaccines. To address this problem an effort is made to develop a subunit vaccine. For this purpose penton base protein of HPS virus was expressed in Escherichia coli and used as subunit vaccine in broilers. Immunogenicity of the recombinant penton base protein and challenge protection test against pathogenic virus demonstrated the ability of recombinant penton base protein to confer (90%) protection. Results suggest that the recombinant penton base protein is a candidate for subunit vaccine against HPS.

Viral infections cause a significant burden on animal health, reducing yields and increasing production costs due to expensive control programs. Vaccination is a vital part of control programs; however, its effectiveness is reduced by the quick evolution of escape viral quasispecies in animal hosts. Existing techniques for studying quasispecies evolution and response to vaccines have severely limited sensitivity and often require prior knowledge of sequence polymorphisms. By generating millions of short reads per run, with no need for culture or cloning, next-generation sequencing (NGS) technologies enable comprehensive identification of viral quasispecies infecting an animal. However, analysis of NGS data is challenging due to the huge amount of data on one hand, and to the short read lengths and high error rates on another. As a consequence, many tools developed for Sanger reads do not work at all or have impractical runtimes when applied to NGS data. Even newly developed algorithms for de novo genome assembly from NGS data are tuned for reconstruction of haploid genomes, and work poorly when the sequenced sample contains a large number of closely related sequences, as is the case in viral quasispecies. To address these shortcomings we have developed computational methods for reconstructing quasispecies sequences and estimate their frequencies from both shotgun and amplicon NGS data. Preliminary analysis of 454 sequencing reads generated from synthetic pools of Infectious Bronchitis Virus (IBV) clones and field isolates collected at various intervals after vaccination with attenuated live IBV vaccine will be presented.

Using peptide nanoparticle technology, we have designed two novel vaccine constructs representing M2e in monomeric (Mono-M2e) and tetrameric (Tetra-M2e) forms. Groups of specific pathogen free (SPF) chickens were immunized intramuscularly with Mono-M2e or Tetra-M2e with and without an adjuvant. Two weeks after the second boost, chickens were challenged with 107.2 EID50 of H5N2 low pathogenicity avian influenza (LPAI) virus. M2e-specific antibody responses to each of the vaccine constructs were tested by ELISA. Vaccinated chickens exhibited increased M2e-specific IgG responses for each of the constructs as compared to a non-vaccinated group. However, the vaccine construct Tetra-M2e elicited a significantly higher antibody response when it was used with an adjuvant. On the other hand, virus neutralization assays indicated that immune protection is not by way of neutralizing antibodies. The level of protection was evaluated using quantitative real time PCR at 4, 6, and 8 days post-challenge with H5N2 LPAI by measuring virus shedding from trachea and cloaca. The Tetra-M2e with adjuvant offered statistically significant (P<0.05) protection against subtype H5N2 LPAI by reduction of the AI virus shedding. The results suggest that the self-assembling polypeptide nanoparticle shows promise as a potential platform for a development of a vaccine against AI.

Background Recent epidemiological investigation of different HA subtypes of avian influenza viruses (AIVs) shows that the H3 subtype is the most predominant among low pathogenic AIVs (LPAIVs), and the seasonal variations in isolation of H3 subtype AIVs are consistent with that of human H3 subtype influenza viruses. Consequently, the development of a rapid, simple, sensitive detection method for H3 subtype AIVs is required. The loop-mediated isothermal amplification (LAMP) assay is a simple, rapid, sensitive and cost-effective nucleic acid amplification method that does not require any specialized equipment. Results A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect the H3 subtype AIVs visually. Specific primer sets target the sequences of the hemagglutinin (HA) gene of H3 subtype AIVs were designed, and assay reaction conditions were optimized. The established assay was performed in a water bath for 50 minutes, and the amplification result was visualized directly as well as under ultraviolet (UV) light reflections. The detection limit of the RT-LAMP assay was 0.1pg total RNA of virus, which was one hundred-fold higher than that of RT-PCR. The results on specificity indicated that the assay had no cross-reactions with other subtype AIVs or avian respiratory pathogens. Furthermore, a total of 176 clinical samples collected from birds at the various live-bird markets (LBMs) were subjected to the H3-subtype-specific RT-LAMP (H3-RT-LAMP). Thirty-eight H3 subtype AIVs were identified from the 176 clinical samples that were consistent with that of virus isolation. Conclusions The newly developed H3-RT-LAMP assay is simple, sensitive, rapid and can identify H3 subtype AIVs visually. Consequently, it will be a very useful screening assay for the surveillance of H3 subtype AIVs in underequipped laboratories as well as in field conditions.

According to our previous study of the M genes of H9N2 avian influenza viruses (AIV) in infected chickens, A/Quail/Hong Kong/G1/97 (G1 97)-like M genes newly emerged in northern and eastern China in addition to the existing A/chicken/Hong Kong/Y280/97 (Y280)-like lineage M genes. To systematically track the genesis and evolution of H9N2 viruses in this region, whole genome sequences of seventeen H9N2 isolates were obtained and their phylogenetic properties were determined. Phylogenetic analysis revealed several newly emerged lineages of gene segments in addition to the Y280-like and A/chicken/Shanghai/F/98(F 98)-like lineages, which are prevailing in northern and eastern China according to previous reports. Reassortments among these gene segments generated five novel genotypes of H9N2 viruses that have not been reported before in China. The emerging genotypes of H9N2 viruses in this region indicate that H9N2 virus genes undergo active evolution, particularly their internal genes, which raises concern for their likely contribution to gene reassortment and production of AIVs with new properties. Our study provides valuable insight into the prevalence of H9N2 viruses in northern and eastern China and demonstrates the need of long-term monitoring of the evolution of H9N2 AIV.

Tuberculosis is one of the important public health problems in Egypt. However, limited information on the Mycobacterium tuberculosis genotypes circulating in Egypt is available. A total of 151 M. tuberculosis strains were characterized by spoligotyping. The results revealed that 74.8% of M. tuberculosis isolates grouped into 13 different clusters, while 25.2% had unique spoligotype patterns. Comparison with an international spoligotyping database (the SITVIT2 database) showed that types SIT53 (T1 variant) and SIT54 (Manu2 variant) were the most common types between cluster groups. In addition, new shared types SIT2977, SIT2978, and SIT2979 were observed. The results identified for the first time an unusually high proportion of ancestral Manu strains of M. tuberculosis from patients in Egypt. The percentage of the Manu clade in this study (27.15%) was significantly higher than its overall representation of 0.4% in the SITVIT2 database. We show that in Egypt tuberculosis is caused by a predominant M. tuberculosis genotype belonging to the ancestral Manu lineage which could be a missing link in the split between ancestral and modern tubercle bacilli during the evolution of M. tuberculosis .

House sparrow (Passer domesticus) is one of the most widely distributed wild birds in China. Five Newcastle disease virus (NDV) strains were isolated from house sparrows living around the poultry farms in southern China. These isolates were characterized by pathogenic assays and phylogenetic analysis. The results showed that all NDV isolates except one were velogenic and virulent for chickens. These four virulent strains for chickens possess the amino acid sequence (112)R/K-R-Q-K/R-R-F(117) in the F(0) cleavage site which is typical of velogenic NDV. Phylogenetic analysis indicated that these isolates belong to genotype VII and were closely related to the strains which were isolated from NDV outbreaks in chickens since 2000. One isolate of NDV from house sparrow belong to genotype II and was proved to be vaccine strain (Chicken/U.S./LaSota/46). The result of this study proved that house sparrow can carry the virulent NDV strains and the same genotype of viruses that are circulating in poultry are existing in house sparrows living around poultry farm in southern China.

Salmonella Enteritidis is a major foodborne pathogen for which chickens serve as reservoir hosts. Reducing Salmonella Enteritidis carriage in chickens would reduce contamination of poultry meat and eggs with this pathogen. We investigated the prophylactic efficacy of feed supplemented with caprylic acid (CA), a natural, generally recognized as safe eight-carbon fatty acid, for reducing Salmonella Enteritidis colonization in chicks. One hundred commercial day-old chicks were randomly divided into five groups of 20 birds each: CA control (no Salmonella Enteritidis, CA), positive control (Salmonella Enteritidis, no CA), negative control (no Salmonella Enteritidis, no CA), and 0.7 or 1% CA. Water and feed were provided ad libitum. On day 8, birds were inoculated with 5.0 log CFU of Salmonella Enteritidis by crop gavage. Six birds from each group were euthanized on days 1, 7, and 10 after challenge, and Salmonella Enteritidis populations in the cecum, small intestine, cloaca, crop, liver, and spleen were enumerated. The study was replicated three times. CA supplementation at 0.7 and 1% consistently decreased Salmonella Enteritidis populations recovered from the treated birds. Salmonella Enteritidis counts in the tissue samples of CA-treated chicks were significantly lower (P0.05) than those of control birds on days 7 and 10 after challenge. Feed intake and body weight did not differ between the groups. Histological examination revealed no pathological changes in the cecum and liver of CA-supplemented birds. The results suggest that prophylactic CA supplementation through feed can reduce Salmonella Enteritidis colonization in day-old chicks and may be a useful treatment for reducing Salmonella Enteritidis carriage in chickens.

Matrix (M) protein genes of 17 H9N2 avian influenza viruses (AIVs) isolated from chickens in northern China during the last 10 years were completely sequenced and phylogenetically analyzed. Homology of nucleotide sequences in the M gene of 17 isolates was 92.7–99.9%. Phylogenetic analysis showed that 11 of the tested M genes belong to the A/chicken/HongKong/Y280/97 (Y280)-like lineage, while the other six belong to the A/Quail/HongKong/G1/97 (G1)-like lineage. This is also the first time that a G1-like M gene of a H9N2 virus was detected in chicken flocks in northern China. These newly appearing changes in M genes may be due to reassortment events of AIVs, or they may have come from the H9N2 strains of southern China which surged in northern China after translocation. An analysis of the viral amino acid sequence of M2 protein has revealed substitution of S31N in two isolates, which is the molecular characterization of amantadine resistance in AIVs. Results of this study suggest that long-term monitoring should be continued to track the transmission and evolution of H9N2 AIVs in chickens in China.