Molecular characterization and phylogenetic analysis of avian influenza H3N8 virus isolated from imported waterfowl in Malaysia

@#Wild aquatic birds are natural reservoirs of influenza A viruses and H3 subtype is one of the most prevalent subtypes in waterfowl. Two H3N8 viruses of low pathogenic avian influenza (LPAI) were isolated via egg inoculation technique from the fecal swab specimens from imported barnacle goose and paradise shelduck in Veterinary Research Institute Ipoh, Malaysia. The full length of eight gene segments of the two viruses were amplified and sequenced with specific primers. The sequences were molecularly characterized, and the sequence identity were assessed with other published sequences. The two viruses are identical and they possess the same amino acid sequences for all the eight gene segments. The viruses were highly similar to the H3 virus from Netherlands and N8 virus from Belgium respectively. Phylogenetic analysis revealed that all the eight gene segments were grouped in the Eurasian lineage, and genetic reassortment may occur between the internal genes of the H3 viruses and other AI subtypes. Though four amino acid substitutions were identified in the hemagglutinin gene, the viruses retained most of the avian-type receptor binding preference. Few amino acid substitutions were observed in all internal genes. Most of the neuraminidase inhibitors and adamantine resistance related mutation were not seen in the viruses. The replicative capacity, cross species transmissibility, and potential zoonotic risk of the viruses are worth further investigation. As H3 virus poses potential threats to both human and animals, and with the increase in the international trade of birds; strict quarantine practice at the entry point and good laboratory diagnostic capabilities is crucial to prevent the introduction of new AI virus into our country.

Identification of Newcastle Disease Virus sub-genotype VII 1.1 isolated from chickens in Sabah, Malaysia

@#Newcastle disease (ND) is an extremely contagious and fatal viral disease causing huge economic losses to the poultry industry. Following recent ND outbreaks in Sabah in commercial poultry and backyard farms, it was speculated that this could be due to a new introduction of Newcastle Disease Virus (NDV) genotype/sub-genotype. Here we report the genetic characterization of NDVs isolated from Sabah during early 2021. All isolates were amplified and sequenced with primers specific to the viral fusion (F) gene using reverse transcription-polymerase chain reaction (RT-PCR). Nucleotide sequence analysis of the F gene showed that all isolates shared similar homology of 99.4% with NDV strain from Iran isolated in 2018. Amino acid sequences of the F protein cleavage site revealed the motif of 112RRQKRF117 indicating all isolates were of virulent strain. Phylogenetic analysis demonstrated that all isolates were clustered under sub-genotype VII 1.1 and clustered together with isolates from Iran (previously known as subgenotype VIIl). The present findings suggested that there is an emerging of a new sub-genotype into the poultry population in Sabah and this sub-genotype has never been reported before in Malaysia. Therefore, transboundary monitoring and continuous surveillance should be implemented for proper control and prevention of the disease. A further molecular epidemiological analysis of NDV is needed to well understand the circulatory patterns of virulent strains of NDV in the country to prevent future outbreaks.

Molecular characterisation of hemagglutinin and neuraminidase genes of the first highly pathogenic Avian Influenza H5N1 2.3.2.1c virus isolated from Sabah, Malaysia

@#Highly Pathogenic Avian Influenza (HPAI) is a highly contagious disease in poultry. The outbreaks can lead to flock mortality up to 100% in two to three days. In July 2018, high mortality in a commercial layer farm in Kauluan village, Sabah was reported. Samples were sent to Veterinary Research Institute Ipoh for diagnosis. Virus isolation and molecular detection is carried out simultaneously. The causative agent was then identified as AI H5N1 virus by real time reverse transcription-polymerase chain reaction (RT-PCR). The virus was then subjected for further nucleotide sequencing of full length hemagglutinin (HA) and neuraminidase (NA) gene. The PQRERRRKR/GLF motif at the HA cleavage site indicated that the isolate was of HPAI virus. Phylogenetic analysis of the HA gene showed that the isolate was belonged to the clade 2.3.2.1c virus. In the HA gene, besides the S133A substitution, the virus possesses conserved amino acid at most of the avian receptor binding sites including the glutamine (Q) and glycine (G) at position 222 and 224 respectively, indicating that the virus retains the avian-type receptor binding preference. As such, the zoonotic potential of the virus was relatively low. On the other hand, though the N154D and T156A substitution were detected in the same gene, the pandemic potential of this Sabah 2.3.2.1c virus is low in the absence of the Q222L, G224S, H103Y, N220K and T315I. A typical 20 amino acid deletion with loss of four corresponding glycosylation sites in the NA stalk region was visible. Though three NA resistance markers were detected, the virus was predicted to be sensitive to NA inhibitor. This is the first HPAI H5N1 outbreak in Sabah. The introduction of this virus into East Malaysia for the first time raised an alert alarm of the future epidemic potential. Strict farm biosecurity, continuous surveillance programme in poultry, wild birds, migratory birds; molecular epidemiology as well as risk assessment for the virus with pandemic potential are needed in dealing with emergence of new influenza virus in the country.

Phylogenetic analysis of nucleoprotein gene of Rabies virus in Malaysia from 2015 to 2018

@#Rabies is a fatal zoonotic disease caused by rabies virus (RABV) and remains a public health problem in Malaysia. Malaysia was declared rabies-free in 2012, however rabies outbreaks occurred at few states in Peninsular Malaysia three years later; and for the first time, in Sarawak (East Malaysia) in 2017 which has caused more than 20 human deaths. This study describes the phylogenetic analysis of the complete nucleoprotein (N) gene of RABV from animal samples in Malaysia from year 2015 to 2018. The N gene of 17 RABVs from Perlis, Kedah and Sarawak were amplified and sequenced. The nucleotide and deduced amino acid similarities of N gene analysis indicated that there is high similarity among the local RABVs. Phylogenetic analysis of the N gene revealed that all Malaysia RABVs belonged to the Asian clade. Among these, RABVs from Peninsular Malaysia were clustered together with RABVs from Thailand, Vietnam and other Southeast Asia countries except Indonesia. However, RABVs from Sarawak were grouped together with Indonesian strains from Kalimantan. Our study provides baseline genetic information of the potential origins of the circulating RABVs in Malaysia. This crucial information helped the authority in policies making and strategies to be taken in outbreak control. Continuous surveillance program to monitor the disease trend, strict border control, vaccination of dog and cat population and public awareness are important steps to control the spread of the RABV.

African swine fever in backyard pigs of Sabah state, East Malaysia, 2021

@#African swine fever (ASF) is a transboundary haemorrhagic viral disease that affected domestic and wild pigs of all ages. The disease is caused by African swine fever virus (ASFV) and was introduced to China in 2018 before spreading rapidly to neighbouring Asian countries. As such, putting countries free from ASF like Malaysia at risk. ASF is highly lethal with no vaccine or treatment available. In February 2021, we confirmed backyard pigs from various locations in Sabah were infected with ASF using real time polymerase chain reaction (realtime PCR). Further characterization of the Sabah ASFVs indicated that they were of p72 genotype II with intergenic region (IGR) variant II that displayed an addition tandem repeat sequence (TRS) insertion, similar to ASFV from Indonesia, Vietnam and China. These results indicate and support the transboundary expansion of a homogenotypic ASFV (p72 genotype II and IGR variant II) in the Europe and Asia-Pacific, emphasizing the need for a holistic international collaboration in control and preventing further spreading of the current ASF pandemic. Importantly, our results informed the first detection and characterization of ASF, a disease previously not detected in Malaysia. This information is crucial for further mitigation and preventive measures.

Molecular Characterization of Avian Infectious Bronchitis Virus Isolated in Malaysia during 2014-2016

@#Avian Infectious Bronchitis (IB) is a highly contagious disease which can cause huge economic losses to the poultry industry. Forty five IB viruses (IBV) were isolated from poultry in Malaysia during 2014-2016. Phylogenetic analysis of the spike glycoprotein 1 (S1) gene revealed that all isolates were clustered into five distinct groups. The predominant type of IBV isolated was QX strains (47%), second was 4/91 type (27%), followed by Malaysian strain MH5365/95 (13%), Massachusetts type (11%) and finally Taiwanese strains (2%). Four types of S1 protein cleavage recognition motifs were found among the isolates which includes HRRRR, RRSRR, RRFRR and RRVRR. To our knowledge, this is the first report describing the motif RRVRR and are unique to Malaysian strains. Six IBVs were grouped in Malaysian MH5365/95 strains. Among these, one isolate was different from others where it only shared 82% identity with MH5365/95 and to others. It formed its own branch in the Malaysian cluster suggesting it may be a variant unique to Malaysia. Alignment analysis of the S1 amino acid sequences indicated that point mutations, insertions and deletions contribute to the divergence of IB variants. This study indicated at least five groups of IBV are circulating in Malaysia with most of the isolates belonged to QX strains. As new IBV variants continue to emerge, further study need to be carried out to determine whether the current available vaccine is able to give protection against the circulating virus.