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Influenza virus A (H5N1) clade 2.3.2.1 has recently caused widespread outbreaks of disease in domestic poultry and wild birds in Eastern Asia. In the current study, the antigenicity and pathogenicity of three clade 2.3.2.1 viruses (Ck/Kr/Gimje/08, Ws/Mongolia/1/09, and Ws/Mongolia/7/10) were investigated in domestic ducks and mice. The H5N1 influenza viruses in this study were antigenically similar to each other (r-values of 0.35-1.4). The three viruses replicated systemically in all tissues tested in domestic ducks, indicating high pathogenicity. However, the viruses produced different clinical signs and mortality rates: Ck/Kr/Gimje/08 and Ws/Mongolia/1/09 resulted in 100% mortality with severe neurological signs, whereas Ws/Mongolia/7/10 resulted in 50% mortality with relatively mild neurological signs. In mice, infection with Ck/Kr/Gimje/08 and Ws/Mongolia/7/10 resulted in weight loss that peaked at 4 days post-infection (22.3% and 20.8%, respectively), same MLD50 (2.2 Log10 EID50) and systemic replication. The three viruses had K deletion at the -2 position of the HA1-connecting peptide (PQRERRRK-R), which is associated with increased virulence in domestic ducks and harbored NA stalk deletion, NS1 deletion and mutation of P42S in NS1, and full length (90aa) in PB1-F2, which confer increased virulence in mice. Our study shows that clade 2.3.2.1 viruses from Korea and Mongolia are antigenically similar and highly pathogenic in both domestic ducks and mice. Moreover, we provide molecular determinants of the clade 2.3.2.1 viruses associated with the pathogenicity in domestic ducks and mice, respectively.

Serial ultrasonographic examinations were performed daily on 9 Miniature Schnauzer bitches from the 15th day of gestation until parturition to determine the time the gestational structures were first detected. The gestational age was timed from the day of ovulation (day 0), which was estimated to occur when the plasma progesterone concentration was >4.0 ng/ml. The gestational length in 9 Miniature Schnauzer bitches was found to be 63.0 +/- 1.7 (range 61-65) days. The initial detection of the fetal and extra-fetal structures were as follows: gestational sac at day 18.0 +/- 0.9 (17-19); zonary placenta in the uterine wall at day 24.9 +/- 1.1 (23-26); yolk sac membrane at day 25.0 +/- 0.9 (24-26); amnionic membrane at day 27.7 +/- 1.0 (26- 29); embryo initial detection at day 22.6 +/- 0.5 (22-23); heartbeat at day 23.4 +/- 0.5 (23-24); fetal movement at day 32.5 +/- 0.8 (32-34); stomach at day 31.2 +/- 1.6 (29-33); urinary bladder at day 32.6 +/- 1.8 (31-35); skeleton at day 34.9 +/- 1.6 (34-38) and kidney at day 42.2 +/- 0.7 (41-43).

Segmental aplasia of the uterine body was diagnosed in a 5-year-old, mixed breed bitch. Abdominal radiography and transabdominal ultrasonography revealed marked dilation of fluid-filled uterine horns with no evidence of a uterine body. Sex hormone assays did not detect the presence of estradiol-17 (β; however, progesterone (2 ng/ml) was found in the serum, indicating anestrus. On gross examination of the reproductive tract, the uterine body was absent, apparently never formed. In its place, a cord-like piece of tissue was identified as an aplastic/dysplastic remnant, connecting the cervix and right uterine horn. The tip of the cord-like piece branched into 5 string-like pieces of tissue, 1 of which was connected to the region dividing the left and right uterine horns. Both the uterine horns were dilated markedly revealing hydrometra. Histologically, uterine body remnant tissues from the endometrium, myometrium, and perimetrium were detected in proximal and distal parts of the uterine body. The string-like piece of tissue connecting the uterine body remnant and the uterine horn consisted of a round cluster of smooth muscle cells surrounding a central core of adipose tissue with blood vessels. It was concluded that the hydrometra observed in both uterine horns was induced by an obstruction resulting from segmental aplasia in the uterine body. This is the first known report of segmental aplasia in the uterine body of a bitch.

The H3 subtype avian influenza virus (AIV) is one of the most frequently isolated subtypes in domestic ducks, live poultry markets, and wild birds in Korea. In 2002–2009, a total of 45 H3 subtype AIVs were isolated from the feces of clinically normal domestic ducks ( n = 28) and wild birds ( n = 17). The most prevalent subtypes in domestic ducks were H3N2 (35.7%), H3N6 (35.7%), H3N8 (25.0%), and H3N1 (3.6%, novel subtype in domestic duck in Korea). In contrast, H3N8 (70.6%) is the most prevalent subtype in wild birds in Korea. In the phylogenetic analysis, HA genes of the Korean H3 AIVs were divided into 3 groups (Korean duck, wild bird 1, and wild bird 2) and all viruses of duck origin except one were clustered in a single group. However, other genes showed extensive diversity and at least 17 genotypes were circulating in domestic ducks in Korea. When the analysis expanded to viruses of wild bird origin, the genetic diversity of Korean H3 AIVs became more complicated. Extensive reassortments may have occurred in H3 subtype influenza viruses in Korea. When we inoculated chickens and ducks with six selected viruses, some of the viruses replicated efficiently without pre-adaptation and shed a significant amount of viruses through oropharyngeal and cloacal routes. This raised concerns that H3 subtype AIV could be a new subtype in chickens in Korea. Continuous surveillance is needed to prepare the advent of a novel subtype AIV in Korea.

On December 7, 2010, H5N1 highly pathogenic avian influenza virus was isolated from a healthy mallard captured at the Mankyung River in South Korea. Phylogenetic analysis showed that this virus was classified into clade 2.3.2 and closely related to H5N1 viruses isolated from wild birds in Mongolia, Russia and China in 2009 and 2010.