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.

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.

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.

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.